Particle on membrane assay system

ABSTRACT

Described herein is an analyte detection device and method related to a portable instrument suitable for point-of-care analyses. In some embodiments, a portable instrument may include a disposable cartridge, an optical detector, a sample collection device and/or sample reservoir, reagent delivery systems, fluid delivery systems, one or more channels, and/or waste reservoirs. Use of a portable instrument may reduce the hazard to an operator by reducing an operator&#39;s contact with a sample for analysis. The device is capable of obtaining diagnostic information using cellular- and/or particle-based analyses and may be used in conjunction with membrane- and/or particle-based analysis cartridges. Analytes, including proteins and cells and/or microbes may be detected using the membrane and/or particle based analysis system.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.60/548,613 entitled “PORTABLE INSTRUMENT FOR MICROARRAY ANALYSIS” filedon Feb. 7, 2004; U.S. Provisional Application No. 60/548,601 entitled“ON-CHIP COMBINATION OF CHEMICAL AND CELLULAR PANELS FOR ANALYSIS OFFLUID SAMPLES” filed on Feb. 7, 2004; and U.S. Provisional ApplicationNo. 60/548,190 entitled “CUSTOMIZED TESTING ENSEMBLES FOR COMPLEX FLUIDANALYSIS USING PORTABLE INTEGRATED MICROFLUIDICS/DETECTING UNITS” filedon Feb. 7, 2004.

BACKGROUND

1. Field of the Invention

The present invention generally relates to a method and device for thedetection of analytes in a fluid. More particularly, the inventionrelates to a portable apparatus for obtaining analytical informationusing both membrane- and particle-based detectors.

2. Description of Related Art

Current methodology used to complete medical diagnostics, environmentalmonitoring, and detection of bioterrorism-related agents often requirelarge and expensive instruments and highly specialized personnel foundonly in certain hospitals, laboratories or government agencies.Furthermore, these instruments are often restricted to a limited numberof applications. For example, in the area of medical diagnostics, eachinstrument is very specialized and designed either to measure proteinlevels or to analyze cellular matter but, typically, may never do both.Additionally, each system is capable of analyzing only a few of therelevant markers of a disease, therefore adding another component to analready tedious and time consuming process that can vary from hours todays. Long delays can be generated between the time of the initialvisit, diagnosis, and administration of treatment, potentially havingdetrimental effects on the prognosis of the disease. Similarly, timelyidentification of an unknown environmental or deliberately introducedcontaminant is crucial. For example, two of the envelopes from the 2001anthrax attacks were processed at a facility that remained open for 9days after the initial contamination, exposing more than 60 million mailitems and more than 2000 employees to Bacillus anthracis spores.

It is therefore desirable that new methods and systems capable ofdiscriminating analytes and/or microbes be developed for health andsafety, environmental, homeland defense, military, medical/clinicaldiagnostic, food/beverage, and chemical processing applications. It isfurther desired that the methods and systems facilitate rapid screeningof analytes and/or microbes to be used as a trigger for more specificand confirmatory testing. It is further desired that sensor arrays bedeveloped that are tailored specifically to serve as efficient microbecollection media.

SUMMARY

In an embodiment, an analyte detection system for both membrane and/orsensor array particle-based measurements may be used to determine thepresence of analytes. In one embodiment, the system may include a samplecollection device, an off-line sample processing unit, a fluid deliverysystem, a disposable cartridge, a cartridge self-positioning system, anoptical platform, electronics, power supplies, computer processor(s),and/or software and firmware. In operation, a sample may be collectedusing the sample collection device. Sample collection devices mayinclude needles, capillary tubes, pipettes, and/or vacutainers. A samplecollection device may be configured to consume a portion of the samplecollection device that contacts a sample. A sample collection device mayinclude a sample pick-up pad configured to receive a sample and deliverthe sample to the cartridge.

In an embodiment, a sample may be transported to a cartridge with thefluid delivery system. A sample may flow from the sample collectiondevice to a sample reservoir in a cartridge. Reagents and/or buffers maybe delivered to the sample reservoir. Reagents may be delivered by areagent delivery system and/or contained in reagent reservoirs, reagentpacks, and/or reagent pads. A sample reservoir may include a mixingchamber where a sample may react with reagents. An actuator coupled tothe cartridge may drive fluid through the cartridge.

A cartridge may include one or more particle-based or particle-basedplatform detection regions and/or membrane based detection regions.Light from an optical platform may pass onto a detection region and adetector in the optical platform may acquire images (e.g., visual orfluorescent) of the sample, and/or of sample-modulated particles. Theimages may be processed and analyzed using software, algorithms, and/orneural networks.

In one embodiment, the system includes the use of defined populations ofassay particles that are chemically sensitized to detect the presence ofa specific analyte in a fluid by binding to the analyte. Chemicallysensitizing a population of particles to detect an analyte may includecoupling a receptor for the analyte to the population of particles. Inan embodiment, receptors for analytes may include antibodies that bindto the analyte. In an embodiment, populations of particles may bedefined by color and/or size. Defining populations of particles by colormay include coupling a fluorescent dye to the population of particles.In an embodiment, analytes may be detected by including a secondchemical that binds to the analyte. In an embodiment, the secondchemical may be a receptor and/or antibody to the analyte. In anembodiment, the second chemical may be defined by a color that isdifferent from the color that defines the population of particles. In anembodiment, the second chemical may be defined by fluorescent dye thatis different from the fluorescent dye that defines the population ofparticles. In an embodiment, detecting an analyte in a fluid may includedetecting two different signals.

In one embodiment, populations of particles may be mechanically capturedon the surface of a filter or membrane-equipped flow cell system. Themembrane-equipped flow cell system may be configured to allow fluid flowthrough the flow cell system and the filter or membrane. In oneembodiment, the membrane-equipped flow cell system may be coupled to anoptical/digital acquisition system that may be configured to allow thevisualization of particles captured thereon. In an embodiment, themembrane-equipped flow cell system coupled to an optical/digitalacquisition system may comprise a device that may facilitate thedigital/optical acquisition of fluorescent signals resulting fromimmunological reactions that take place on the surface of themembrane-captured particles.

In an embodiment, a detecting an analyte in a fluid may include forminga mixture of size- and color-coded particles with the fluid. Theparticles may be coupled to a receptor that interacts with the analyte.In an embodiment, the particle/fluid mixture may be passed across aporous membrane equipped in an analyte detection device. In anembodiment, an analyte detection device may include a flow cell system.In an embodiment, the analyte detection device may be configured tocapture the particles on the porous membrane. In an embodiment, theanalyte detection device may be configured to visualize the particlescaptured on the membrane. In an embodiment, detecting the analyte mayinclude detecting spectroscopic signals from the particles captured onthe membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

Features and advantages of the methods and apparatus of the presentinvention will be more fully appreciated by reference to the followingdetailed description of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

FIG. 1A depicts an exploded view of a membrane based flow sensor;

FIG. 1B depicts a schematic of an embodiment of a microchip;

FIG. 1C depicts a schematic of an embodiment of a microchip with alocking mechanism;

FIG. 1D depicts a schematic of an embodiment of positions of cavities ina microchip;

FIG. 1E depicts a schematic of an embodiment of a pattern of cavities ina microchip;

FIG. 1F depicts a schematic of an embodiment of an alternate pattern ofcavities in a microchip;

FIG. 2 depicts an embodiment of a membrane based flow sensor disposed ina housing;

FIG. 3 depicts a schematic diagram of an analyte detection system inflow-through mode;

FIG. 4 depicts a schematic diagram of an analyte detection system inlateral flow mode;

FIG. 5 depicts a schematic diagram of an analyte detection system inback-flush mode;

FIG. 6 depicts a flow chart of a method of collecting samples;

FIG. 7 depicts a graph of calibration bead intensity vs. amount ofsample added;

FIGS. 8A-8F depict a method of analysis of particles captured by amembrane;

FIG. 9 depicts a schematic diagram of a membrane based analyte detectionsystem that includes a sensor array detection device;

FIG. 10 depicts porous particles;

FIGS. 11A-D depicts a schematic diagram of a bead optimization method;

FIG. 12 depicts a schematic diagram of a flow cytometer;

FIGS. 13A-B depict a schematic diagram of a multi-layer artificialneural network;

FIG. 14 depicts a schematic diagram of the preparation of multi-shellparticles;

FIG. 15 depicts a diagram of the shrinking core model for multi-shellparticles in a monoanalyte system;

FIGS. 16A-D depict graphical representations of multi-componentfingerprint responses yielded by functional multi-shell particles uponthe introduction of an analyte;

FIG. 17 depicts a schematic diagram of the preparation of multi-shellparticles having a common core with different outer layer ligands;

FIG. 18 depicts plots of t_(L) values for three different multi-shellparticle types vs. metal concentration;

FIG. 19 depicts plots of red, blue and green absorbance of a multi-shellparticle vs. time for multiple analytes;

FIG. 20 depicts a diagram of the shrinking core model for multi-shellparticles in a bianalyte system;

FIG. 21A-C depicts plots of red, blue and green Absorbance vs. timeplots for an EDTA-ALZC particle;

FIG. 22A-D depicts an array of graphs showing the responses of anEDTA-ALZC particle to binary mixtures of Ca(NO₃)₂ and MgCl₂;

FIG. 23A-B depict plots of a particles primary (23A) and secondary (23B)delays vs. Mg²⁺ and Ca²⁺ concentration;

FIG. 24 depicts breakthrough curves for a Cd and Hg mixture on cysteineand histidine conjugated particles;

FIGS. 25A-B depict the detection of Hepatitis B HbsAg in the presence ofHIV gp41/120 and Influenza A in an embodiment of a sensor array system;

FIG. 26 depict the detection of CRP in an embodiment of a sensor arraysystem;

FIG. 27 depicts the dosage response of CRP levels in an embodiment of asensor array system;

FIGS. 28A-D depict the multi-analyte detection of CRP and IL-6 in anembodiment of a sensor array system;

FIG. 29 depicts the regeneration of receptor particles in an embodimentof a sensor array system;

FIG. 30 depicts a schematic diagram of a device for membrane and/orparticle-based analyte detection;

FIGS. 31A-D depict schematic diagrams of sample collection systems;

FIG. 32A depicts a schematic diagram of a detection system withactuators;

FIG. 32B depicts an embodiment of an actuator;

FIG. 32C depicts an embodiment of a channel coupled to a samplecartridge;

FIGS. 33A-C depict schematic diagrams of disposable sample cartridges;

FIG. 33D depicts an exploded view of a cartridge with a reagent capsule;

FIG. 33E depicts schematic diagram of a cartridge with a reagentcapsule;

FIG. 34 depicts an embodiment of a fluid delivery system that includes asample probe;

FIG. 35 depicts an embodiment of a fluid delivery system that includes afour-way fluidics interface;

FIGS. 36A-B depict an embodiment of a fluid delivery system thatincludes a three-way fluidics interface;

FIG. 37 depicts a schematic diagram of a cartridge self-positioningsystem;

FIG. 38A depicts a schematic diagram of an optical platform;

FIG. 38B depicts an embodiment of a light emitting diode assembly;

FIG. 38C depicts an exploded view of an embodiment of light emittingdiode assembly;

FIG. 39 depicts a schematic diagram of an optical platform that includesthree light sources;

FIG. 40 depicts a schematic diagram of an optical platform that includestwo light sources;

FIG. 41 depicts an optical platform that includes two laser lightsources;

FIG. 42 depicts a schematic diagram of an optical platform that includesa single optical fiber microlens;

FIG. 43 depicts a schematic diagram of an optical platform that includesmultiple optical fiber microlenses;

FIG. 44A-B depicts a schematic diagram of an optical platform thatincludes a multiple optical fibers to conduct signals to an analysisdevice;

FIG. 45 depicts an analyte detection device that includes both aparticle-based detection system and a membrane-based detection system;

FIG. 46 depicts an exploded view of a portion of a detection systemsupport system;

FIG. 47 depicts an analyte detection device that includes both aparticle-based detection system and a membrane-based detection systemhaving an external pump;

FIG. 48 depicts an embodiment of a disposable cartridge for use in thedetection of analytes;

FIG. 49 depicts a roller system configured to force liquid from one ormore blister packs disposed in a cartridge;

FIG. 50 depicts an embodiment of a disposable cartridge for use in thedetection of analytes having input ports configured to connect tostandard sampling equipment;

FIGS. 51A-C depict a sequence of steps for reacting a sample with areagent in a mixing chamber;

FIGS. 52A-C depict a series of schematic diagrams showing the operationof a cartridge;

FIG. 53 depicts a schematic drawing of an alternate embodiment of acartridge;

FIGS. 54A-C depict different embodiments of inlet and outlet channels ina cartridge;

FIGS. 55A-D depict different embodiments of channels for deliveringfluids within a cartridge;

FIGS. 56A-B depicts different embodiments of cartridges that include atrap;

FIGS. 57A-C depict different embodiments of cartridges that include afluidics interface;

FIGS. 58A-B depict an embodiment of polystyrene particle types definedby size and by fluorescence signal intensity;

FIGS. 59A-C depicts an embodiment of the particle on membrane assaysystem;

FIGS. 60A-D depicts the detection of TNF-α in a fluid according to anembodiment;

FIG. 61 depicts a dose response to TNF-α according to an embodiment;

FIG. 62 depicts an embodiment of a cartridge that includes a sensorarray;

FIG. 63 depicts an embodiment of a portion of the cartridge depicted inFIG. 63;

FIG. 64 depicts an embodiment of a reagent reservoir and reagent pack inthe cartridge depicted in FIG. 63;

FIG. 65A depicts an embodiment of a blister pack containing reagents;

FIG. 65B depicts a cross-sectional view of a blister of a blister pack;

FIG. 66 depicts an embodiment of valves positioned in the cartridgedepicted in FIG. 63;

FIGS. 67A-67C depict views of the operation of a pinch valve;

FIG. 68 depicts a cross-sectional view of a pinch valve;

FIG. 69A depicts an exploded view of an embodiment of a cartridge thatincludes a sensor array;

FIG. 69B depicts a top view of the cartridge of FIG. 69A;

FIG. 69C depicts a perspective view of an embodiment of the cartridge ofFIG. 69A;

FIG. 69D depicts a bottom view of an embodiment of the cartridge of FIG.69A;

FIG. 70 depicts an exploded side view of an embodiment of a cartridge;

FIG. 71 depicts a side view of an embodiment of a cartridge;

FIG. 72A depicts an exploded view of an alternate embodiment of acartridge that includes a sensor array;

FIG. 72B depicts an embodiment of an arrangement of valves in thecartridge of FIG. 72A;

FIG. 73A depicts an exploded view of an embodiment of the cartridgedepicted in FIG. 72A as sample is introduced in the cartridge;

FIG. 73B depicts an embodiment of an arrangement of valves in acartridge as sample is introduced in the cartridge;

FIG. 74A depicts an exploded view of an embodiment of the cartridgedepicted in FIG. 72A after the sample is introduced into the channel;

FIG. 74B depicts an embodiment of an arrangement of valves in acartridge after the sample is introduced into the channel;

FIG. 75A depicts an exploded view of an embodiment of the cartridge ofFIG. 72A in which a reservoir is being actuated;

FIG. 75B depicts an embodiment of an arrangement of valves in acartridge that allows a sample to be pushed towards a detection regionusing buffer released from a reservoir;

FIG. 76 depicts an embodiment of buffer pushing sample towards adetection region;

FIG. 77 depicts an embodiment of buffer pushing sample towards adetection region;

FIG. 78A depicts an exploded view of an embodiment of a cartridge; and

FIG. 78B depicts an embodiment of an arrangement of valves in acartridge.

DETAILED DESCRIPTION

Herein we describe a system and method for the simultaneous analysis ofa fluid containing multiple analytes. The system may generate patternsthat are diagnostic for both individual analytes and mixtures of theanalytes. The system, in some embodiments, is made of a combination ofchemically sensitive particles, formed in an ordered array, capable ofsimultaneously detecting many different kinds of analytes rapidly. Anaspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

Details regarding analyte detection systems can be found in thefollowing U.S. patents and patent applications, all of which areincorporated herein by reference: U.S. patent application Ser. No.09/287,248 entitled “Fluid Based Analysis of Multiple Analytes by aSensor Array”; U.S. Pat. No. 6,680,206 entitled “Sensor Arrays for theMeasurement and Identification of Multiple Analytes in Solutions”; U.S.Pat. No. 6,602,702 entitled “Detection System Based on an AnalyteReactive Particle”; U.S. Pat. No. 6,589,779 entitled “General SignalingProtocols for Chemical Receptors in Immobilized Matrices”; U.S. patentapplication Ser. No. 09/616,731 entitled “Method and Apparatus for theDelivery of Samples to a Chemical Sensor Array”; U.S. patent applicationSer. No. 09/775,342 entitled “Magnetic-Based Placement and Retention ofSensor Elements in a Sensor Array” (Published as U.S. Publication No.:2002-0160363-A1); U.S. patent application Ser. No. 09/775,340 entitled“Method and System for Collecting and Transmitting Chemical Information”(Published as U.S. Publication No.: 2002-0064422-A1); U.S. patentapplication Ser. No. 09/775,344 entitled “System and Method for theAnalysis of Bodily Fluids” (Published as U.S. Publication No.:2004-0053322); U.S. Pat. No. 6,649,403 entitled “Method of Preparing aSensor Array”; U.S. patent application Ser. No. 09/775,048 entitled“System for Transferring Fluid Samples Through A Sensor Array”(Published as U.S. Publication No.: 2002-0045272-A1); U.S. patentapplication Ser. No. 09/775,343 entitled “Portable Sensor Array System”(Published as U.S. Publication No.: 2003-0186228-A1); U.S. patentapplication Ser. No. 10/072,800 entitled “Method and Apparatus for theConfinement of Materials in a Micromachined Chemical Sensor Array”(Published as U.S. Publication No.: 2002-0197622-A1); and U.S. patentapplication Ser. No. 10/427,744 entitled “Method and System for theDetection of Cardiac Risk Factors” (Published as U.S. Publication No.:2004-0029259-A1).

In another embodiment, a sensor array system may be a membrane basedflow sensor. A membrane based flow sensor may be configured toaccommodate the capture of microbes and/or cells with a filter that isplaced within a fluidics device. Microbes and/or cells, whose size islarger than the pores of the filter, are captured in the flow cellassembly. The captured microbes and/or cells may be analyzed directly ormay be treated with visualization compounds.

A variety of microbes may be captured and analyzed using a membranebased flow sensor as described herein. As used herein, “microbe” refersto any microorganism, including but not limited to, a bacteria, spore,protozoan, yeast, virus, and algae. Some microbes that are of particularinterested for detection include a variety of toxic bacteria. Examplesof bacteria that may be detected using a membrane based flow sensorinclude, but are not limited to Escherichia coli O157:H7,Cryptosporidium, Vibrio cholerae, Shigella, Legionnella, Lysteria,Bacillus globigii, and Bacillus anthracis (anthrax). Viruses may also bedetected using a membrane, including the HIV virus.

Shown in FIG. 1A is an exploded view of a membrane based flow sensor100. Flow sensor 100 includes a membrane 110 that is sandwiched betweenat least two members 140 and 150. Members 140 and 150 are configured toallow fluid to flow to and through membrane 110. Members 140 and 150 arealso configured to allow detection of analytes, after the analytes havebeen captured on membrane 110. A variety of different materials may beused for membrane 110, including, but not limited to, Nuclepore®track-etched membranes, nitrocellulose, nylon, and cellulose acetate.Generally, the material used for membrane 110 should have resistance tonon-specific binding of antibodies and stains used during thevisualization and detection processes. Additionally, membrane 110 iscomposed of a material that is inert to a variety of reagents, buffers,and solvents. Membrane 110 may include a plurality of sub-micron poresthat are fairly evenly distributed. The use of membranes having an evendistribution of pores allows better control of fluid flow and control ofthe isolation of analytes.

Members 140 and 150 are composed of a material that is substantiallytransparent to wavelengths of light that are used to perform the analytedetection. For example, if the analyte detection method requires the useof ultraviolet light, member 140 should be composed of a material thatis substantially transparent to ultraviolet light. Member 140 may becomposed of any suitable material meeting the criteria of the detectionmethod. A transparent material that may be used to form member 140includes, but is not limited to, glass, quartz glass, and polymers suchas acrylate polymers (e.g., polymethylmethacrylate). In someembodiments, both top member 140 and bottom member 150 are composed oftransparent materials. The use of transparent materials for the topmember and the bottom member allow detection to be performed through themembrane based flow sensor.

As shown in FIG. 1A, membrane 110 is sandwiched between top member 140and bottom member 150. Bottom member 150 and/or top member 140 mayinclude indentations configured to hold a membrane. For example, in FIG.1A, bottom member 150 includes an indentation 152 that is configured toreceive membrane 110, along with any other accompanying pieces that areused to support or seal membrane 110. Indentations or cavities may beetched into top member 140 and/or bottom member 150 using standardetching techniques.

Referring to FIG. 1A, bottom member 150 includes a first indentation152, which is configured to receive a membrane support 130. Bottommember also includes a second indentation 154. Second indentation isconfigured such that membrane support 130 is inhibited from entering thesecond indentation. Second indentation may include a ridge disposed nearthe membrane support 130 such that membrane support 130 rests upon theridge. Alternatively, as depicted in FIG. 1A, second indentation may beto may have a size that is smaller than the size of membrane support130. In either case, when assembled, membrane support 130 is inhibitedfrom entering second indentation 154, thus creating a cavity undermembrane support 130. Cavity 154 may be used to collect fluids that passthrough the membrane support 130 prior to exiting the system.

Membrane support 130 is configured to provide support to membrane 110during use. Membrane support 130 may be formed from a porous materialthat allows fluid to pass through the membrane support. The pores ofmembrane support 130 should have a size that allows fluid to passthrough membrane support 130 at a speed that is equal to or greater thanthe speed that fluid passes through membrane 110. In one embodiment,pores of membrane support 130 are larger than pores in membrane 110. Thepores, however, cannot be too large. One function of membrane support130 is to provide support to membrane 110. Therefore, pores in membranesupport 130 should be sufficiently small enough to inhibit sagging ofmembrane 110 during use. Membrane support 130 may be formed of a varietyof materials including, but not limited to, polymeric materials, metals,and glass. In one embodiment, a polymeric material (e.g., celconacrylic) may serve as a material for membrane support 130. Additionally,membrane support 130 helps to keep the membrane planar during use.Keeping the membrane planar simplifies detection of the analytes byallowing the capture and detection of the analytes on a single focalplane. Membrane 110, as described above, may rest upon membrane support130 when the membrane based flow sensor 100 is assembled. In someembodiments, a gasket 120, may be positioned on top of membrane 110. Agasket may be used to provide a fluid resistant seal between members 130and 140 and membrane 110. Gasket may inhibit the leakage of fluid fromthe system during use.

Top member 140 may include a fluid inlet 160. Fluids for analysis may beintroduced into device 100 via fluid inlet 160. Fluid inlet 160 may passthrough a portion of top member 140. In some embodiments, a channel 162may be formed in top member 140 such that tubing 164 may be insertedinto channel 162. Channel 162 may turn near the center of the top memberto deliver the fluids to an upper surface of membrane 110.

Bottom member 150 may include a fluid outlet 170. Fluids that areintroduced into the device 100 via fluid inlet 160 pass through topmember 140 and through membrane 110. The fluids are then collected incavity 154. A fluid outlet 170 may pass through a portion of bottommember 150. In some embodiments, a channel 172 may be formed in bottommember 150 such that tubing 174 may be inserted into channel 172.Channel 172 may be positioned to receive fluids that are collected incavity 154 during use.

Optionally, a washing fluid outlet 180 may be formed in top member 140.Washing fluid outlet 180 is configured to receive fluids that passthrough or over membrane 110 during a washing operation. Washing fluidoutlet 180 may pass through a portion of top member 140. In someembodiments, a channel 182 may be formed in top member 140 such thattubing 184 may be inserted into channel 182. Channel 182 may bepositioned to receive fluids that are used to wash membrane 110 duringuse.

Membrane 110 is selected from a material capable of filtering theanalytes of interest from a fluid stream. For examples, if microbesrepresent the analyte of interest, the filter should be capable ofremoving microbes from a fluid stream. A suitable membrane may include aplurality of pores that have a size significantly less than the size ofthe analyte of interest. For airborne toxic microbes (e.g., anthrax),the membrane may be configured to capture microbes that have a diameterof greater than about 1 μm. It is believed that microbes that have adiameter of less than about 1 μm are very difficult to generate in largequantities, and if the organisms are viable, environmental stresses tendto interfere with the action of the microbes due to the high surfacearea/mass ratio. Membranes may be formed from a variety of materialsknown in the art. In one embodiment, membrane 110 may be a track-etchedNuclepore™ polycarbonate membrane. A Nuclepore membrane is availablefrom Whatman plc. Membrane 110 may be about 5-10 microns in thickness.Membrane 110 includes a plurality of pores. Pores may range from about0.2 μm in diameter up to about 12 μm in diameter to capture potentiallydangerous microbes.

In some embodiments, a membrane may include a plastic and/or metallicmaterial with a high density of pores. A membrane may be made of amaterial which is substantially non-reflective and/or substantiallyinhibits emission in the UV-vis range. For example, materials that amembrane may be formed from include, but are not limited to,polymethylmethacrylate (PMMA); polycarbonate (PC); Delrin® (commerciallyavailable from DuPont); titanium; silicon; silicon nitride; and/orcombinations thereof. A membrane support may be formed from variousmaterials including, but not limited to, polymethylmethacrylate (PMMA);polycarbonate (PC); Delrin®; titanium; silicon; silicon nitride; and/orcombinations thereof.

In some embodiments, a membrane and a membrane support may be combinedto create a monolithic microchip. A monolithic microchip may be madethrough various techniques such as LIGA fabrication, which may allowdesign and fabrication of high aspect ratio features; injection molding;through bundled optical fibers assemblies; and/or LASER etching. Amicrochip may be substantially circular, substantially rectangular,substantially square, substantially triangular, and/or have an irregularshape.

FIG. 1B depicts a schematic of an embodiment of a membrane. In FIG. 1B,holes in the membrane are drawn larger than their actual size forclarity. A membrane may be configured to have pore dimensions toaccommodate a variety of applications including, but not limited to,capturing microorganisms and/or particles in the range of about 100 nmto about 1 mm in size. A membrane may have a thickness, t; a diameter,D; holes with a diameter, d; and/or a density of holes on the microchip,ρ. In an embodiment, a specific set of parameters for t, D, d, and ρmaybe used for a specific application. Various applications may includevarious definitions of specific sets of parameters for ρ, D, d, and t.In an embodiment, a membrane may have pores ranging from about 100 nm toabout 1 mm and/or a thickness ranging from about 1 to about 5000microns. A membrane may have a thickness of from about 1 μm to about2000 μm.

As depicted in FIG. 1C, a membrane may include a locking mechanism. Alocking mechanism 190 for a microchip 191 may be substantially circular,substantially rectangular, substantially square, substantiallytriangular, and/or have an irregular shape. A locking mechanism mayinhibit insertion of an incorrect microchip in a system. For example, ananalyte detection system may be capable of receiving a membrane that hasthe correct corresponding locking mechanism. If the locking mechanism isnot of the proper shape and/or orientation, the membrane will not fitinto the system. In this way only the proper membrane may be insertedinto the system. A locking mechanism may also facilitate secureplacement of a membrane in a desired location. Using a locking mechanismmay facilitate consistent placement of a membrane in the same locationin the system. A locking mechanism 190 may be positioned on a sideand/or bottom of a membrane 191. It should be understood that particlebased sensor arrays, as described herein, may also include a similarlocking mechanism for ensure insertion of the correct sensor array inthe correct position.

FIGS. 1D-E depict patterns of cavities or holes in a membrane. Holes,openings, or cavities in a membrane may be positioned in a pattern,randomly positioned, and/or orderly positioned. Patterns created byholes in a membrane may cover the whole membrane or may be restricted togiven areas of the membrane. In some embodiments, a membrane 191 mayhave independent compartments 192 separated by walls 193 or ridges.Walls in a membrane may be configured to have various geometries andheight. A wall may define multiple compartments. In certain embodiments,compartments of a membrane may be connected to either the same drainand/or separate independent drains. Using compartments may allowdelivery of fractions of a given sample to different compartments. Usingcompartments may also allow one sample to be delivered sequentially tovarious independent areas of a membrane. In another embodiment,different samples may be delivered to different compartments allowinganalysis of multiple samples using a single membrane.

In some embodiments, the independent areas or compartments of a membranemay be characterized as having pores of different sizes. Pore sizes in acompartment may be configured to accommodate applications such as, butnot limited to, sequential sieving, cell sorting, bead sorting, andmultiplexing based on size. In an embodiment, a membrane or variouscompartments of a membrane may be configured to include one or morecavities. Cavities may include particles that interact with an analyteto produce a detectable signal. The cavities may be square-basedpyramidal or conical and/or may have a shape to accommodate beads ofdifferent sizes.

FIG. 1F depicts a schematic of an embodiment of a membrane that includesa particle-based sensor array. A membrane 191 may include a combinationof cavities 194 capable of receiving one or more particles and holes 195that may be used to capture analytes by filtration. Cavities 194,capable of receiving particles or beads, may be in a differentcompartment 192 from holes 195 in a membrane. Walls 193 on a membrane191 may separate compartments including cavities 194 with particles fromcompartments that include holes 195. In some embodiments, a combinationof particle wells and holes in different compartments may allowsimultaneous on-chip capture and detection of cells and protein analysisof complex fluids (i.e., blood, urine, CSF, etc.). In certainembodiments, a microchip may include one or more calibration aids suchas, but not limited to, beads, fluorescent elements, size reference,and/or topographical points of reference.

FIG. 2 depicts an embodiment of a membrane based flow sensor disposed inhousing 200. Top member 140, gasket 120, membrane 110, membrane support130, and bottom member 150 may be assembled and placed inside housing200. Housing 200 may encompass membrane based fluid sensor. A cap 210may be used to retain membrane based fluid sensor within housing 200.Cap 210 may include a window to allow viewing of membrane 110. Whenpositioned within housing 200, fluid inlet 160, fluid outlet 170 andwashing fluid outlet 180 extend from housing 200 to allow easy access tothe membrane based fluid sensor 100.

A schematic of a complete membrane based analysis system is shown inFIG. 3. Analysis system includes a plurality of pumps (p₁, p₂, p₃ andp₄). Pumps are configured to deliver samples (p₁), visualizationreagents (p₂ and p₃) and membrane washing fluids (p₄) to the membranebased fluid sensor 100 during use. Reagents, washing fluids, andvisualization agents are passed through pre-filters (f₁, f₂, f₃, and f₄)before the fluids are sent to membrane based fluid sensor 100.Pre-filters are used to screen out large particulate matter that mayclog membrane 110. The nature and pore size of each pre-filter may beoptimized in order to satisfy efficient capture of large dust particlesor particulate matter aggregates while resisting clogging. Pre-filter f1is configured to filter samples before the samples reach the membranebased fluid sensor 100. Pre-filter f1 is configured to allow the analyteof interest to pass through while inhibiting some of the particles thatare not related to the analyte of interest. For example, spores, whosesize is smaller than the pores of the pre-filter f₁, are passed throughthe pre-filter and captured in the membrane based fluid sensor 100.After passing through pre-filters f₁-f₄, fluids are passed through amanifold. In some embodiments, membrane based fluid sensor 100 includesa single input line. The manifold couples the different fluid lines tothe single input line of the membrane based fluid sensor 100.

After passing through the manifold, fluids are introduced into fluidinlet of the membrane based fluid sensor 100. At appropriate times, adetector 250 is used to determine if any analytes have been captured bythe membrane based fluid sensor 100. As depicted in FIG. 3, a detectormay be placed over a portion of membrane based fluid sensor 100 suchthat the detector may capture an image of the membrane. For example,detector may be placed such that images of the membrane may be takenthrough a window in the membrane based fluid sensor 100. Detector 250may be used to acquire an image of the particulate matter captured onmembrane 110. Image acquisition may include generating a “digital map”of the image. In an embodiment, detector 250 may include a highsensitivity CCD array. The CCD arrays may be interfaced with filters,light sources, fluid delivery, so as to create a functional sensorarray. Data acquisition and handling may be performed with existing CCDtechnology. In some embodiments, the light is broken down intothree-color components, red, green and blue. Evaluation of the opticalchanges may be completed by visual inspection (e.g., with a microscope)or by use of a microprocessor (“CPU”) coupled to the detector. Forfluorescence measurements, a filter may be placed between detector 250and membrane 110 to remove the excitation wavelength. The microprocessormay also be used to control pumps and valves as depicted in FIG. 3.

The analyte detection system may be operated in different modes based onwhich valves are opened and closed. A configuration of a system in a“flow through” mode is depicted in FIG. 3. In this mode, fluid is passedfrom the manifold to the membrane based fluid sensor 100 to allowcapture of analytes or the addition of development agents. Fluids foranalysis may be introduced into membrane based fluid sensor 100 viafluid inlet 160. During a “flow through” operation, valve V₁ is placedin a closed position to inhibit the flow of fluid through wash fluidoutlet 180. The fluids may, therefore, be forced to pass throughmembrane based fluid sensor 100 exit the sensor via fluid outlet 170.Valve V₂ is placed in an open position to allow the flow of fluid to thewaste receptacle. Valve V₃ is placed in a closed position to inhibit theflow of fluid into the wash fluid supply line.

The analyte detection system may also be operated in a “lateral membranewash” mode, as depicted in FIG. 4. In this mode, the membrane is clearedby the passage of a fluid across the collection surface of the membrane.This allows the membrane to be reused for subsequent testing. Fluids forwashing the membrane may be introduced into sensor 100 via fluid inlet160. During a “lateral membrane wash” operation, outlet valves V₂ and V₃are placed in a closed position to inhibit the flow of fluid throughfluid outlet 170. The closure of outlet valves V₂ and V₃ also inhibitsthe flow of fluid through the membrane of sensor 100. The fluidsentering sensor 100 may, therefore, be forced to exit sensor 100 throughwashing fluid outlet 180. Valve V₂ is placed in an open position toallow the flow of fluid through washing fluid outlet 180 and into thewaster receptacle. Since fluid is inhibited from flowing through themembrane, any analytes and other particles collected by the membrane maybe “washed” from the membrane to allow further use.

The analyte detection system may also be operated in a “backwash” mode,as depicted in FIG. 5. During a backwash operation, fluid outlet 170 isused to introduce a fluid into the analyte detection system, while washfluid outlet 180 is used to allow the fluid to exit the device. This“reverse” flow of fluid through the cell allows the membrane to becleared. In an embodiment, valves may be configured as depicted FIG. 5,with the washing fluid being introduced through fluid outlet 170.Specifically, valves V1 and V3 are open, while valve V2 is closed.

Either a lateral membrane wash or a back flush treatment may be used toclear analytes and other particles from a membrane. Both methods ofclearing the membrane surface may be enhanced by the use of ultrasoundor mechanical agitation. During use, analytes in the fluid sample aretrapped by the membrane since the analytes are bigger than the openingsin the membrane. The analytes tend to be randomly distributed across themembrane after use. Analytes that occupy positions on the membrane thatare between the positions of pores may be harder to remove them analytesthat are position on or proximate to a pore in the membrane. Analytesthat occupy positions on the membrane that is between the positions ofpores may be more difficult to remove, since the force of the backwashfluid may not contact the analytes. During backwash and lateral washoperations, removal of trapped analytes may be enhanced by the use ofultrasound of mechanical agitation. Both methods cause the analytes tomove across the membrane surface, increasing the chances that theanalyte will encounter a column of washing fluid passing through one ofthe pores.

Analyte detection system may be used to determine the presence ofanalytes in a fluid system. One embodiment of a process for determininganalytes in a fluid sample is depicted in the flow chart of FIG. 7.Prior to the analysis of any samples, a background sample may becollected and analyzed. Solid analytes are typically collected andstored in a liquid fluid. The liquid fluid that is used to prepare thesamples, may be analyzed to determine if any analytes are present in thefluid. In one embodiment, a sample of the liquid fluid used to collectthe solid analytes is introduced into an analyte detection device todetermine the background “noise” contributed by the fluid. Any particlescollected by the membrane during the background collection are viewed todetermine the level of particulate matter in the liquid fluid. In someembodiments, particles collected by the membrane during the collectionstage may be treated with a visualization agent to determine if anyanalytes are present in the liquid fluid. The information collected fromthe background check may be used during the analysis of collectedsamples to reduce false positive indications.

After collection of the background sample, the membrane may be clearedusing either a back flush wash or a lateral wash, as described herein.After clearing the membrane, the system may be used to analyze samplesfor solid analytes (e.g., microbes). As used herein the term “microbes”refers to a variety of living organisms including bacteria, spores,viruses, and protozoa. As the collected sample is passed through theporous membrane, the porous membrane traps any particles that have asize that is greater than the size of the pores in the porous membrane.Collection of particles may be continued for a predetermined time, oruntil all of the collected sample has been passed through the membrane.

After collection, the particles collected by the membrane may beanalyzed using a detector. In some embodiments, the detector may be acamera that will capture an image of the membrane. For example, adetector may be a CCD camera. Analysis of the particles captured by themembrane may be performed by analyzing the size and/or shape of theparticles. By comparing the size and/or shape of the particles capturedby the membrane to the size and shape of known particles the presence ofa predetermined analyte may be indicated. Alternatively, microbeanalytes will react to a variety of visualization agents (e.g., coloredand fluorescent dyes). In one embodiment, the detection of microbeanalytes may be aided by the staining of the microbe with avisualization agent. The visualization agent will induce a known colorchange or impart fluorescence to a microbe. In an embodiment, particlescaptured by the membrane are stained and the particles analyzed using anappropriate detector. The presence of particles that have theappropriate color and/or fluorescence may indicate the presence of theanalyte being tested. Typically, non-microbe particles (e.g., dust) willnot undergo the same color and/or fluorescent changes that microbes willwhen treated with the visualization agent. The visualization agent mayinclude a “cocktail” mixture of semi-specific dyes, which may bedesigned to mark microbes of interest. Selection of the mixture may bebased on the capacity of the dye chromophore to create an opticalfingerprint that can be recognized by a detector and associated imagingsoftware as being associated with specific pathogenic bacteria orspores, while at the same time distinguishing from the signal exhibitedby dust and other background particulate matter.

The analysis of the particles may indicate that an analyte of interestis present in the sample. In this case, the particles may be flushedfrom the membrane and sent out of the system for further testing.Further testing may include techniques such as cultures or ELISAtechniques that may allow more accurate determination of the specificanalytes present. Alternatively, the particles may be sent to a sensorarray, as described herein, for further testing. If no significantamounts of analytes are found on the membrane, the membrane may bewashed and other samples analyzed.

In an embodiment, user-defined threshold criteria may be established toindicate a probability that one or more specific microbes are present onthe membrane. The criteria may be based on one or more of a variety ofcharacteristics of the image. In some embodiments, the criteria may bebased on pixel or color fingerprints established in advance for specificmicrobes. The characteristics that may be used include, but are notlimited to, the size, shape, or color of portions of matter on theimage, the aggregate area represented by the matter, or the totalfluorescent intensity of the matter. In an embodiment, the system mayimplement an automated counting procedure developed for one or morepathogenic and non-pathogenic bacteria.

In an embodiment, the membrane system may include a computer system (notshown). Computer system may include one or more software applicationsexecutable to process a digital map of the image generated usingdetector. For example, a software application available on the computersystem may be used to compare the test image to a pre-defined opticalfingerprint. Alternatively, a software application available on computersystem may be used to determine if a count exceeds a pre-definedthreshold limit.

A detector may be used to acquire an image of the analytes and otherparticulate matter captured on a membrane. Microbes may collect on amembrane along with dust and other particulate matter and be captured inan image produced from a detector. The image acquired by the detectormay be analyzed based on a pre-established criteria. A positive resultmay indicate the presence of a microbe. The test criteria may be basedon a variety of characteristics of the image, including, but not limitedto, the size, shape, aspect ratio, or color of a portion or portions ofthe image. Applying test criteria may allow microbes to be distinguishedfrom dust and other particulate matter. During analysis, the flow ofsample through from a fluid delivery system may be continued.

In some embodiments, a positive result may create a presumption that thefluid contains a particular analyte. If the image yields a positiveresult with respect to the test criteria, a sample of the fluid may besubjected to a confirmatory or specific testing. On the other hand, ifthe image yields a negative result with respect to the test criteria,membrane may be rinsed and the preceding method may be carried out forfluid from another sample.

During analyte testing a sample may be introduced into the analytedetection device. A trigger parameter may be measured to determine whento introduce the visualization agent into the analyte detection device.Measurement of the trigger parameter may be continuous or may beinitiated by a user. Alternatively, the stain may be introduced into theanalyte detection device immediately after the sample is introduced.

In one embodiment, the trigger parameter may be the time elapsed sinceinitiation of introducing the fluid into an analyte detection device ata controlled flow rate. For example, the stain may be introduced 20seconds after initiation of introducing the fluid sample into an analytedetection device at a flow rate of 1 milliliter per minute. In anotherembodiment, the trigger parameter may be the pressure drop across themembrane. The pressure drop across the membrane may be determined usinga pressure transducer located on either side of the membrane.

In another embodiment, the trigger parameter may be the autofluorescenceof analytes captured by the membrane. A detector may be switched onuntil a pre-defined level of signal from the autofluorescence of theanalytes has been reached. In still another embodiment, filteringsoftware may be used to create a data map of the autofluorescence of thematter on the membrane that excludes any pixels that contain color in ablue or red spectral range. The data map may be used to compute a valuefor particles that are autofluorescent only in the “pure green” portionof the visible spectrum.

In some embodiments, a presumptive positive result may be inferred ifthe trigger parameter exceeds a certain value without applying a stain.For example, a presumptive positive result may be inferred where theautofluorescence value is more than twice the value that would indicateapplication of a stain. In such a case, the application of a stain maybe dispensed with and a confirmatory test may be conducted for thesample.

If the value of the trigger parameter is less than would indicateproceeding directly to the confirmatory test, but exceeds the valueestablished to trigger the application of a stain, then a stain may beintroduced into an analyte detection device.

Collecting a sample of a fluid may include gathering a sample from asolid, liquid, or gas. In some embodiments, the sample may be derivedfrom collecting air from a target environment in an aerosol form, thenconverting aerosol into a hydrosol. For example, particles from 500liters of an air sample may be collected deposited into about 0.5milliliters of liquid. U.S. Pat. No. 6,217,636 to McFarland, entitled“TRANSPIRATED WALL AEROSOL COLLECTION SYSTEM AND METHOD,” which isincorporated herein by reference as if fully set forth herein, describesa system for collecting particulate matter from a gas flow into a liquidusing a porous wall.

In one embodiment, a system as described above, may be used to determinethe presence of anthrax spores or bacteria. Collection of air samples ina potentially contaminated area may be concentrated in a fluid sampleusing an aerosol collector. The fluid sample may be passed through amembrane based detector system as described herein. The membrane baseddetection system may collect any particle collected by the aerosolcollector. The particles collected may be treated with a visualizationagent that includes stains that are specific for anthrax bacteria. Suchvisualization agents are know to one of ordinary skill in the art. Thepresence of particles that exhibit the appropriate color/fluorescencemay indicate that anthrax is presence. The indication of anthrax may befurther determined by additional confirmation testing.

Experimental

Flow Cell

The flow cell assembly was created from a 3-piece stainless steel cellholder consisting of a base, a support and a screw-on cap. Two circularpolymethylmethacrylate (PMMA) inserts house the nuclepore® membrane.These two PMMA inserts have been drilled along their edge and side toallow for passage of the fluid to and from the chip through stainlesssteel tubing (#304-H-19.5, Microgroup, Medway, Mass.). The tubes, whichwere fixed with epoxy glue in the drilled PMMA inserts had an outerdiameter of 0.039″ (19.5 gauge), and a 0.0255-0.0285″ inner-diameter.The basic components of the flow cell are two disc-shaped PMMA“inserts”. The bottom PMMA insert is modified in order to feature adrain and to contain a plastic screen disc (Celcon acrylic) that acts asa support for the filter. Each insert features a length of stainlesssteel tubing, which enters a hole in the side of the PMMA disk. The topinsert also features an additional outlet which is used whenregeneration of the filter is needed. Silicone tubing is snapped on thestainless steel tubing, and as such is readily compatible with a widerange of fluidic accessories (i.e., pumps, valves, etc.) and solvents.The flow cell was shown to be resistant to leaks and pressures generatedby flow rates as high as 20 mL/min.

Fluid Delivery, Optical Instrumentation and Software

The complete analysis system shown in FIGS. 3, 4, and 5 includes afluidics system composed of four peristaltic pumps (p₁, p₂, p₃, and p₄),dedicated to the delivery of the analyte collected from the air,antibody, wash buffer to the flow cell, and clean-up off the flow cellin the regeneration mode. Its integrated software was used to assurefluid delivery to the chip, and accommodate wash cycles through theproper use of valves. The sample, antibody, PBS, and regeneration linesare also filtered (pre-filters f₁, f₂, f₃, and f₄) to screen out largeparticulate matter. Pre-filter f₁ is a nuclepore® filter with a poresize of 5 μm Pre-filters f₂, f₃, f₄ are 0.4 μm nuclepore® filters.Spores which size is smaller than the pores of pre-filter f, are passedthrough the filter and captured in the analysis flow cell, positioned onthe motorized stage of a modified compound BX2 Olympus microscope. Themicroscope is equipped with various objectives, optical filters, and acharged-coupled device (CCD) camera which operation can be automated.

A Mercury lamp was used as the light source. Fluorescence images shownin this report were obtained with a FITC filter cube(fluoroisothiocyanate, 480 nm excitation, 505 long pass beam splitterdichroic mirror, and 535±25 nm emission), and captured by a DVC 1312C(Digital Video Company, Austin, Tex.) charge-coupled device (CCD)mounted on the microscope and interfaced to Image Pro Plus 4.0 software(Media Cybernetics). Areas of interest of the images of the array forwere selected in an automated fashion and used to extract numericalvalues of the red, green, and blue (RGB) pixel intensities.

Reagents

Phosphate buffer saline (PBS), pH 7.4, was purchased from Pierce (#28374, 0.008M Na₃PO₄, 0.14M NaCl, 0.01M KCl). The content of thepre-weighted pack was dissolved in 500 mL dI water. After completedissolution, the buffer solution was filtered using a 60 mL disposablesyringe (Becton Dickinson #309654) and a 0.2 mm pore size syringe filter(Whatman 25 mm, 0.2 mm Polyethersulfone (PES) filters #6896-2502).Polyoxyethylene-Sorbitan Monolaurate (Tween-20) and Bovine SerumAlbumine (BSA) were purchased from Sigma (# P-1379, and # A-0281). Theanti-bg antibody was generously given to us by Tetracore, and taggedwith a fluorophore. The naked Antibody was labeled according to theprotocol described in the Alexa Fluor® 488 Protein labeling kit fromMolecular Probes (# A-10235), and stored at 4° C. The finalconcentration of the labeled anti-bg was 0.5 mg/mL. When prepared forthe assay the antibody was diluted 50 times in a filtered (3 mLDisposable Syringes from Becton Dickinson # 309574; Syringe Filters fromPall Gelman 13 mm, 0.2 μm Acrodisc CR Polytetrafluoroethylene PTFE #4423) solution of 1% BSA/PBS (0.01 g of BSA per mL of PBS). The sporepreparations were given to us by Edgewood/Dugway Proving Grounds. Fortheir evaluation, the spores were membered onto Petri dishes and grownwith Luria Bertani plating medium. The medium is composed of BactoTryptone, Bacto Yeast Extract, Agar Technical purchased from Difco (#211705, # 212750, # 281230 respectively), and NaCl purchased from EM (#SX0420-1). Distilled Water, de-ionized with a Barnstead Nanopure Columnwas autoclaved for 30 min. at 121° C. to sterilize it.

Polymer Microsphere Solutions

The fluorescent polymer green microspheres were purchased from DukeScientific Corporation (Palo Alto, Calif.). A bead stock solution wasprepared by diluting several drops of the original bead solution in 500mL of DI water. A bright line counting chamber, or hemacytometer(Hausser Scientific, Horsham, Pa.) was used to determine the exactconcentration of this solution. The concentration of a solution istypically obtained from the average of several measurements following awell established protocol. The concentration of our stock solution wasfound to be 1,883,750 beads/mL±8539 or a relative standard deviation of0.45%. For the solutions used in FIG. 3 and FIG. 4, we used a 1 to 50dilution of the stock solution, and added 50 μL, 100 μL, 150 μL, 200 μL,and 250 μL of that solution to the same flow cell, and captured imagesat different magnifications.

Bg Spore Solutions Preparation

A 1 mg/mL spore stock solution (A) was prepared in sterile water bysuspending x mg of spores in x mL of sterile water. Solutions B, C, D,E, F, G, H and I with respective concentrations of 10e-1, 10e-2, 10e-3,10e-4, 10e-5, 10e-6, 10e-7, and 10e-8 mg/mL were obtained by serialdilution of the stock solution A.

Bg Spore Solutions Characterization

The concentration of spores per mg of preparation was evaluated bygrowing colonies in a Luria Bertani culture media and expressed inColonies Formation Unit (CFU) per mg of spore. 15 g of Bacto Tryptone,7.5 g of Bacto Yeast Extract and 15 g of NaCl were dissolved in 1.5 L ofsterile water. The pH was adjusted to 7.6 (Fisher Accumet pH meter 620)using a 0.1N NaOH solution. 22.5 g of Agar technical were then added tothe preparation. The solution was heated in a microwave to allowcompleted dissolution and autoclaved for 30 min. at 121° C. Aftercooling, the media was poured in disposable sterile culture members(Fisherbrand #08-757-12). The members were left until the media hadtotally solidified and then wrapped with Parafilm for storage.

The number of CFU per mg of the Bg spore Preparation was evaluated asfollows: 100 μL of solutions A to I were grown in the culture media at37° C. for 24 hrs. After incubation, colonies had grown enough to becounted. Only members with a statistical number of colonies (between 30and 300) were used to calculate the number of CFU per mg of sporepreparation. Solutions A to E had too numerous counts (TNC) and solutionH and I had not enough counts (under 30). In addition, sterile water wasalso used as a negative control and gave 0 CFU. The averageconcentration was determined from the remaining members as 3×10⁸ CFU/mgof spore preparation.

Assay Optimization

The specificity of the Tetracore antibody for Bg spores was confirmedfirst by in-tube reactions and subsequent evaluation with fluorescencemicroscopy of stained spores on glass slides. The same antibody was thenemployed for the detection of Bg spores captured on the filter membraneof our system. A series of tests were performed in order to identifythose conditions resulting in the highest signal to noise ratio for thison-line assay. Parameters evaluated included: a) the effect ofpre-treating the system's tubing and filter membrane with BSA (i.e.blocking of non-specific binding sites for the detecting antibody), b)varying the rate (i.e. flow rate) of antibody introduction to the flowcell, c) varying the antibody concentration, d) varying the incubationtime of the antibody with Bg spores, e) identifying the optimal exposuretime for image capture, and f) comparison of uni-directional mode ofantibody flow to the cell versus re-circulation. Our studies revealedthat blocking the system's tubing and the flow cell's filter membranewith BSA offered no significant advantage for the assay in terms ofreducing the non-specific signal. Nonetheless, we found that when 1% BSAwas included in the antibody solution, the Bg-specific signal wasenhanced, resulting in a higher signal to noise ratio and, therefore, amore sensitive assay. An incubation time of Bg spores for five minuteswith 1.5 mL of Bg-specific antibody at 10 μg/mL, which was introduced inthe flow cell in uni-directional mode (i.e. in to flow cell and out towaste) at 0.3 mL/min were identified as the optimal conditions for theassay.

Our studies also showed that re-circulation of the antibody did notoffer any advantage in terms of shortening the assay time or decreasingits detection limit. Even though such an approach could potentiallyreduce the amount of antibody utilized in the assay, we decided againstit because prolonged re-circulation of the antibody was associated withits precipitation. As expected, precipitated antibody could be capturedby the membrane and thus result in an increase of the non-specificsignal. On the contrary, there was very little precipitation of thedetecting antibody when delivered in unidirectional mode. We equippedthe system with a 0.4 μm pre-filter, which prevented any precipitatedantibody from reaching the analysis flow cell. This approach resulted ina much cleaner assay.

Finally, we determined that the appropriate exposure time for capturingthe final images for this assay was 184 ms. This exposure time was suchthat it produced the strongest Bg-specific signal and the weakestbackground, non-specific signal resulting from contaminants such asdust, irrelevant unstained bacteria and fluorescent paper fibers thatcould potentially be found in the system.

Dose Response Curve

To establish the standard curve, the spore solutions were prepared in asimilar fashion as described previously with PBS instead of sterilewater. Briefly, a 1 mg/mL (or 3×10⁸ CFU/mL) spore stock solution A wasprepared by suspending 1 mg of spores in 1 mL of PBS. Solutions B, C, D,E, F and G were obtained from stock solution A by serial dilution,resulting in concentrations of 3×10⁸, 3×10⁷, 3×10⁶, 3×10⁵, 3×10⁴, 3×10³,3×10² CFU/mL respectively for solutions A, B, C, D, E, F, and G. Theseconcentrations cover the range from 1 ng/mL to 1 mg/mL. For eachsolution, an assay was conducted through execution of the followingsteps. The solution is introduced through pump 1 for 60 s at a flow rateof 1 mL/mn, and followed by a 60 s PBS wash through pump 2 with the sameflow rate. The antibody is then slowly (0.3 mL/min) passed through pump3 to the flow cell. A final 90 s wash ensures the removal of any unboundor non-specifically attached antibody. The background signal wasevaluated through five independent measurements of the signal obtainedfrom the passage of antibody in five different spore-free flow cells.The limit of detection was chosen as 3 times the standard deviationobtained from the average of these five measurements. The calibrationcurve was built from the measurement of four different spore solutionsaccounting for 900, 3000, 9000, and 30000 spores. A fluorescentmicrograph of the signal remaining after the final wash was recorded andthe signal expressed as the density of green intensity per pixel. Theaverage green density per pixel was plotted as a function of spore countdetermining a limit of detection of 900 spores.

Electron Microscopy

Correlative light and electron microscopy was accomplished by placing a5 μL aliquot of antibody-stained spores on a Formvar-coated TEM grid(Maxtaform H2 finder grids, Ted Pella, Inc). Due to the thick walls ofthe spores, it was possible to avoid more complex dehydration regimensand simply allow the spore suspension to air dry. After a suitable areawas located and photographed with fluorescence microscopy, the grid wasplaced in a Philips 420 TEM and the same grid square was photographed.The grid was then affixed to an aluminum stub with carbon tape andsputter-coated with gold palladium. Using a Leo 0.1530 SEM, images werecaptured from the area of interest.

Bead Tests

In order to determine the functionality as well as the analyticalvalidity of our system, we tested our integrated system with 2.3 μm and1 μm fluorescent polymer microspheres (purchased from Duke ScientificCorporation). The size of these particles was chosen to best simulatepopulations of spores and bacteria. The calibration curves displayingthe average density per pixel as a function of added volume are shown inFIG. 7. Examination of these graphs reveals that the linearity of thedetected response is not affected by the magnification. However, asexpected, the slope of the regression lines increases with increasingmagnification as the signal from the beads is brighter at highmagnification. Many factors, such as the size and brightness of thebacteria or spores, the total area of the membrane exposed to theanalyte, the field of view, dictate the experimental parameters to beused. Because they are very homogeneous in size and intensity, polymericbeads represent an ideal calibrator and simulant for spores. However,the actual size of spores is slightly smaller than that of the beadsthat were used, and the signal produced from a singlespore-antibody-fluorophore complex is much less intense than that of themicrospheres. Additionally, fluidics concerns prevent us from using toosmall a filter area, because the internal pressure is greatly raised asthe fluid is forced through a dramatically reduced number of pores.Because the magnification does not change the linearity of thecalibration curves as shown in FIG. 7, and in order to accommodate asustained flow through the flow cell, an objective of 5×, for a totalmagnification of 100× was chosen for the assay.

Spores and Bacteria

To illustrate the capabilities of our detection system, we targetedBacillus globigii (Bg), a commonly used non-pathogenic simulant forBacillus anthracis (Ba). An immuno-assay was created, based on thecapture of Bg spores and their interaction with a Bg-specific antibodyresulting in the formation of an immuno-complex. The effect of possibleinterferences in the assay was also tested with a variety of speciessuch as yeast, talc powder, and other species of Bacillus as will bediscussed later in this report. In FIG. 5 is shown a fluorescentmicrograph of Bg spores stained with an Alexa® 488-labeled anti-Bacillusglobigii antibody. The schematic of the immuno-complex is shown in theinset. In order to demonstrate the specificity of the interaction of theanti-Bg antibody with the Bg spores, we conducted some correlationstudies between the fluorescence micrographs and the images obtainedfrom transmission electron microscopy (TEM) and scanning electronmicroscopy (SEM). An aliquot of immuno-labeled Bg was placed on aFormvar-coated TEM finder grid, and epifluorescence micrographs wereobtained at various magnifications. The grids were then imaged withtransmission electron microscopy (TEM), after which they were coatedwith gold palladium and imaged with scanning electron microscopy (SEM).As illustrated by the correspondence of the fluorescence signal with theposition of the spores, the finder grid made it possible tounequivocally locate the same area in each instrument, clearlyindicating that the fluorescence signal arises from the Alexa®488-tagged antibody that is specifically binding to the Bg spores.Fluorescence micrographs obtained at a total magnification of ˜400× areshown in order to better represent this correlation. However, thecorrelation of the fluorescence signal from spores with TEM or SEMmicrographs is also established with magnification as low as ˜100×.

To determine the limit of detection of our system, we conducted adose-dependence study. Solutions of spores were prepared by serialdilution of a stock spore solution, presuming that 1 mg of dry sporesper mL yields 10⁸ spores per mL. Following the flow cell experiments,aliquots of the spore solutions were analyzed to determine the exactspore concentration in terms of colony forming units per mL (CFU). Thebackground was determined as the signal obtained after passage of theantibody through a blank filter and subsequent rinsing with PBS. Inorder to assess the limit of detection, the standard deviation wascalculated from the average of 5 such measurements of the background.The limit of detection was established to be 900 spores.

As the internal volume of the flow cell is very small, it is necessaryto flush out all contaminants in order to avoid clogging of the membranefilter. Of particular importance for these studies is the control ofdust, commonly and abundantly found in the postal environment. SEMstudies (not shown) have demonstrated that the dust produced throughtransport, manipulation, and processing of postal mail, contains fibers,debris, and various kinds of bacteria. Most significantly, dust containsa large number of particles with a wide size distribution encompassingthe size range of the biological agents of interest. Furthermore, manyof the dust components exhibit autofluorescence, due to the use offluorescent brighteners and inks in the paper and document industries.Many of the trigger systems currently used in military type detectorsrepose on size selection principles such as Aerodynamic Particle Sizing(APS) or Flow Cytometry (FC), and for the reasons exposed previously, donot appear as the ideal trigger systems. Our system was tested in ablind study against triggering by yeast, talc, and powdered detergents.The rate of success was 100% as no false positive was generated. Anothermajor potential problem arising from accumulation of dust in our systemis clogging of the nuclepore® filter. We have conducted studies whichshowed that failure of the flow cell operation occurs only after 60 mgof dust are passed through, building a pressure greater than 60 psi,corresponding to 400 hours of postal operation, assuming that theconcentration of dust reaching the flow cell is an average 6.2 μg/L.However, this result is widely dependent on the efficiency of theaerosol system and it is based on the assumption that the aerosolcollection system has a built-in capability of discarding at least 95%of dust particles of 10 μm or higher. In these conditions, even thoughthe accumulation of dust in the flow cell is inevitable in the long run,the device still exhibits a lifetime well above that desired formilitary applications. Additionally, we have shown that it is possibleto regenerate the flow cell and extend its lifetime by flushing out upto 99% of the dust, spores, or debris accumulated on the filter. Thisfunction can easily be implemented through the use of an additionaloutlet within the top insert of the flow cell, and implementation of anautomated flush protocol. A combined method of sonication, backflow, andlateral flow is used to eliminate unwanted material from the membrane.This allows for extended operation of the detection system without theattention of a technician. The removal of spore-sized (0.93 μm)fluorescent polymer microspheres from the membrane surface during fiveconsecutive trials was performed. Surface plots in column i representsthe initial loading of the membrane in the flow cell. Efficiencies of95%, 98%, 99%, 99%, 99% is reached, respectively, for the five trials.

Pixel Analysis Methods for Detection of Microbes

In some embodiments, pixel analysis methods may be used in the analysisof an image of a fluid or captured matter. For example, pixel analysismay be used to discriminate microbes from dust and other particulatematter captured on a membrane. Pixel analysis may include analyzingcharacteristics of an image to determine whether a microbe is present inthe imaged fluid.

Pixel analysis may be based on characteristics including, but notlimited to, the size, shape, color, and intensity ratios of an image orportions of an image. As an example, the total area that emits light inan image may be used to conduct analysis. As another example, the greenfluorescent intensity of an image may be used to conduct analysis. In anembodiment, an “optical fingerprint” for a specific microbe or set ofmicrobes may be established for use in pixel analysis. In someembodiments, pixel analysis may be based on ratios between values, suchas an aspect ratio of an element of matter captured on an image. Inother embodiments, pixel analysis may be based on threshold values.

During use, a visualization agent may cause different particles to emitdifferent wavelengths of light depending on the nature of the particle.When the particles are analyzed with a camera, a user may be able todetermine if a particular analyte is present based on the color of theparticle. For example, a green particle may indicate the presence of ananalyte of interest. Any other colored particles may not be of interestto a user. While a person may be able to discern between colors, it isdesirable for a computer system to also be able to discern differentcolors from a membrane sample. Many detectors can only discern specificcolors when analyzing an image. For example, many CCD detectors can onlydiscern red, blue and green colors. Thus, a CCD detector may not be ableto discern the difference between a particle that emits both blue andgreen light and a particle that just emits green light, although thecolor difference may be apparent to a person using the system. Toovercome this problem a method of subtracting out particles having the“wrong” color may be used.

In some embodiments, pixels of an image that do not fall within a colorrange specified by a user may be discarded from the image. In oneembodiment, a fluid may be stained to cause a microbe of interest toemit light in only the green portion of the visible spectrum. Bycontrast, dust and other particles contained in the fluid may emit lightin combinations of green, blue, and red portions of the visible spectrumin the presence of the stain. To isolate the portion of the image thatrepresents only the microbe of interest, binary masks may be created toeliminate light emissions caused by non-microbial matter from the image.

Such a method is depicted in FIGS. 8A-F. FIG. 8A shows an image of allparticles captured by a membrane. For purposes of this example,particles 500, having the no fill pattern, exhibit a green color;particles having a fill pattern identical to the fill pattern ofparticle 510 have a red color; particles having the a fill patternidentical to the fill pattern of particle 520 have both green and bluelight absorption; particles having a fill pattern identical to the fillpattern of particle 530 have both red and blue light absorption; andparticles having a fill pattern identical to the fill pattern ofparticle 540 have a blue color. It should be understood that these colorassignments are for illustrative purposes only. In the current example,the goal of the analysis is to find all of the green particles.

One method of finding the green particles is to use a filter that willallow only particles that are green are shown. FIG. 8B depict theparticles that would remain if such a filter is used. All of theparticles shown in FIG. 8B have a green light absorption, however, notall of the particles that are depicted in FIG. 8B would exhibit a greencolor only. Particles 520 absorb both green and blue light. Since thedetector can't differentiate between the two types of particles, a falsepositive may result.

To compensate for this phenomena, images of particles that absorb blueand red are also analyzed using appropriate filters. By creating masksof which particles exhibit blue and red absorption, a process ofelimination may be used to determine how many green particles arepresent. In an embodiment, an image is then captured of only theparticles that exhibit color in the red portion of the spectrum (SeeFIG. 8C). The image of “red” particles is used to create a mask that maybe compared to the full spectrum view of the particles. Since theanalytes of interest only exhibit color in the green portion of thespectrum, any particle with color in the red portion of the spectrum maybe removed from the original image. FIG. 8D shows the original image butwith the particles that appear in the red portion of the spectrumsubtracted from the image. The remaining particles are potentialparticles that may be the analyte of interest.

In a second iteration, FIG. 8E shows a binary mask that may be used tomask any pixels that include a blue component. An image is captured ofonly the particles that exhibit color in the blue portion of thespectrum (See FIG. 8E). The image of “blue” particles is used to createa mask that may be compared to the full spectrum view of the particles.Since the analytes of interest only exhibit color in the green portionof the spectrum, any particle with color in the blue portion of thespectrum may be removed from the original image. FIG. 8F shows theoriginal image but with the red binary mask and blue binary mask appliedso that pixels including a red or blue component are excluded. Theparticles that remain in the image are thus particles that only exhibita green color. Thus, the method may be used to produce an image thatincludes only “pure green” pixels. Such an image may be analyzed todetect the presence of a microbe by eliminating particles that are notrelevant. It should be understood that while the above recited exampleis directed to determining the presence of green particles it should beunderstood that the process can be modified to determine blue particlesonly, red particles only, or particles that exhibit combinations ofcolors (e.g., red and blue, red and green, blue and green, or red, blueand green).

In some embodiments, pixel analysis may be used in combination with themembrane method for detecting a microbe described herein. Pixel analysismay be conducted either before or after the application of a stain. Inan embodiment, pixel analysis may be used to determine when to apply astain.

After an analyte of interest is detected using a membrane baseddetection device further testing may be performed to identify theanalyte. In one example, the particles captured by the membrane may betransferred to a sensor array as described in any of the patents andpatent applications previously listed.

FIG. 9 depicts a system in which a particle sensor array detector 600 iscoupled to a membrane analyte detection device 100. Membrane basedanalyte detection device may be part of an analyte detection system aspreviously described. After a sample is passed through a membrane, theparticles collected by the membrane may be subjected to an additionaltest to further identify the analytes. In one embodiment, the analytesmay be washed from the surface o the membrane and transferred to asensor based analyte detection system, as described in any of thepreviously referenced patent applications. The analytes extracted fromthe sample may react with beads that are placed in a sensor array. Thereaction of the analytes with the sensor array beads may allowconfirmation (or further identification) of the analytes. Methods ofdetecting microbes using a sensor array system are described in furtherdetail in the above-referenced patent applications.

Many microbes may not react with a bead of a sensor array. Largemicrobes may be unable to make proper contact with the bead andtherefore are not detected by the bead. In one embodiment, the microbesare subjected to a treatment that allows better detection by a beadbased detection system In one embodiment, the particles may be subjectedto lysis conditions. Lysis of microbes will cause the disintegration ordissolution of the microbe. For bacteria, lysis may be induced bytreatment with an alkali base or by use of enzymes. Lysis of thebacteria allows portions of the material contained by the bacteria to bereleased and analyzed. Typically, either proteins or nucleic acidsreleased from the bacteria may be analyzed.

Microbes such as bacteria, spores, and protozoa in a fluid may becaptured in the macropores of the beads. In some embodiments, receptors,including, but not limited to, selective antibodies or semi-selectiveligands such as lectins, may be coupled to a particle in an internalpore region of the particle to create a selective bead. Suitablereceptors may be selected using the methods described herein. In someembodiments, a visualization antibody may be introduced that may couplewith the captured analyte. The visual antibody may yield a colorimetricor fluorescence signature that can be recorded by the CCD detector. Insome embodiments, a series of selective and semi-selective beads may beused in conjunction with the sensor array system described herein.

In an embodiment, an agent that is known to bind or interact with amicrobe may be introduced into a fluid prior to the time that themicrobes are placed in proximity with a sensor array. Such agents mayhave characteristics that facilitate capture of a microbe by a particlein the sensor array.

Macroporous Particles

In an embodiment, a particle having macropores may be formed of agarose.A depiction of such a particle is shown in FIG. 10. A particle may be inthe form of a spherical bead. The particle may include a plurality ofmacropores on its surface and interior.

In an embodiment, agarose may be used as a starting material for amacroporous particle because it is biocompatible and may be capable ofinteracting with biomolecules and living organisms. Activated agarosemay demonstrate an affinity interaction with bacteria andmicroorganisms. To facilitate this interaction, specific properties onparticles may be used to target specific microorganisms or cells.Processed agarose, in which sulfate groups have been eliminated from theagarose chain, may consist of an uncharged hydrophilic matrix withprimary and secondary alcohols that can be used for activation andattachment. For example, the chemical surface of particles may bemodified by oxidizing adjacent diols into aldehyde groups. Using sodiummeta-periodate (NaIO₄) aliphatic aldehydes may be obtained that can beused in reductive amination coupling procedures.

In an embodiment, macroporous particles may be formed by suspensionpolymerization using a gel. Size, shape, and uniformity of the particlemay depend on the hydrophilic or hydrophobic additives used to stabilizethe emulsion. Pore size may be determined by agarose concentration ofthe gel. Mechanical properties, such as gel strength, may be affected bythe molecular weight of the agarose. In one embodiment, suspensionpolymerization may be accomplished using a biphasic system containingthe agarose monomer and emulsion stabilizers. A dispersion of ahydrophilic emulsifier (such as TWEEN 85) in cyclohexane may be added toa stirring aqueous solution of agarose at 60° C. for 5 min to produce anoil-in-water emulsion. Fine particles of agarose stabilized by theemulsifier may be formed in this step. Next, a solution of a hydrophobicemulsifier (such as SPAN 85) may be added to the first emulsion forminga water-in-oil emulsion. Afterwards, the water-in-emulsion may be cooledto room temperature. Polymeric particles may appear at about 40° C. Theaggregation of droplets, which may be referred to as flocculation, mayform a matrix with oil droplets that will form pores or conduits in thebeads. The particles may be washed with distilled water and alcohol,sized with industrial sieves, and preserved in water.

Emulsifiers added to the hydrophilic and/or hydrophilic phases and theconcentration of the agarose solution may influence the quality of thebeads. Additionally, mixing speed, nature of the agitation, andtemperature during the preparation process may be important. Thestability of the solutions may depend on the selected emulsifiers andthe solvents used.

A particle may be of a substantially spherical shape. Particles withspherical geometry may enhance the available area for surfaceinteraction with the analytes. Creating pores within the particles mayalso increase surface area. Particles may have large connecting flowpores in addition to normal diffusion pores. A macroporous particle mayhave improved mass transfer properties compared to a non-macroporousparticle.

A particle may have a diameter of between about 250-300 microns.Macropores in a particle may be less than about 1 micron. Different poresizes and shapes may allow for the entrapment and detection of a varietyof analytes, including, but not limited to, cells, bacteria, DNAoligomers, proteins/antibodies, and small molecules.

An alternative process to suspension polymerization may be the use of afoaming agent to vary the porosity of the particles. For example, thedecomposition of azides or carbonates during polymerization may allowincorporation of nitrogen or carbon dioxide “bubbles” into theparticles. Because the gelling point for agarose is 40° C., thedecomposition reaction should be performed at low temperatures.

Another alternative to suspension polymerization may be the use ofmolecular imprinting. The imprinting of particles may occur bynon-covalent and covalent methods. Non-covalent imprinting may be basedon non-covalent interactions such hydrogen bonds, ionic bonds, and Vander Waals forces between functional monomer and a temmember. Thestability of the monomer-temmember complex prior to polymerization maydepend on the affinity constants between the temmember and thefunctional monomers. In the covalent method, the bonds formed betweenthe functional monomer and the temmember may be cleaved once thepolymerized matrix is obtained.

Within the covalent and non-covalent based approaches, there may bedifferent methods for making molecular imprinted polymers. One approachmay involve grinding the imprinted polymer to reduce their size toapproximately 25 μm and expose the imprinted surfaces. Anothertechnique, which may be referred to as ‘surface temmemberpolymerization,’ uses water and oil. In this technique, thewater-soluble temmember may interact with the functional monomer at thewater-oil interface. The complex monomer-temmember in the organic phasemay be polymerized yielding a polymer-imprinted surface. This techniquemay allow the imprinting of water-soluble substances like zinc ions.

Other methodologies for imprinting polymers may be suitable. Molecularimprinting on microgel spheres may be a convenient procedure forimprinting agarose because the imprinted gel does not need to be reducedin size by grinding as in conventional molecular imprinting. Discreteimprinted microgels and imprinted microspheres may be synthesized bycross-linking polymerization of the monomer in the presence of thetemmember, a process known as “precipitation polymerization.”

Surface temmember polymerization and precipitation polymerization may besuitable alternative techniques to chemical surface modification ofregular particles. Both techniques may be suitable for imprintingagarose with such temmembers as bacterial spores. A chromatographycolumn mounted with imprinted beads may be a fast method for evaluatingthe efficacy of the imprinted beads. For example, bacteria may bere-bound, exposed to the fluorescent calcium-sensitive indicator knownas calcein, and detected by fluorescence spectroscopy.

Molecular imprinting may allow the exploitation of organisms asreactors. The pores in the particle may facilitate feeding of entrappedmicroorganism reactants and cause them to produce a desired product.Molecular imprinting may be used for encapsulating bacteria such as theRhizobium organisms into agarose. These bacteria may convert nitrogenfrom the atmosphere into ammonia. By “feeding” these bacteria nitrogen,ammonia may be produced. The pores encapsulating the bacteria may retainan imprint of the organism for morphologic studies of the bacteria'ssurface.

High-performance liquid chromatography and fluorescent assays may be avaluable tool for studying the molecularly imprinted polymers. Thefluorescent dye acridine orange may stain agarose beads so they may bemorphologically analyzed with confocal scanning laser microscopy. Othermorphological studies include atomic force microscopy, scanning electronmicroscopy, and microtome techniques. Characterization of the surfacearea of the beads may be achieved by measuring the adsorption isothermand using the Brunauer, Emmet, and Teller equation.

In some embodiments, the surface of a particle may be chemicallymodified. In other embodiments, chemical functionality, including, butnot limited to non-specific (i.e., functional groups) and highlyspecific (i.e., bio-ligands such as antibodies) may be localized intothe confines of the pore region. Chemical functionality may facilitatethe entrapment of a variety of analytes.

In an embodiment, a particle may include a receptor that includes aparticular metal. The metal may be capable of binding a material that ischaracteristic of a particular analyte. For example, a particle may beformed that includes terbium (III). Terbium (III) forms a luminescentcomplex with dipicolinic acid, a substance unique to spores.

EXAMPLE

Macroporous beads were prepared using the method for biphasic suspensionpolymerization method described herein. The beads so obtained wereanalyzed using light and fluorescence microscopy. The transparency ofthe agarose beads permitted the visualization of the fluorescent beadsin different sections of the agarose beads. The presence of pores wasconfirmed by adding 1 μm fluorescent beads. Using light and fluorescencemicroscopy, the presence of conduits could not be conclusivelydetermined. The beads accumulated into voids present in the bead,probably the ends of conduits.

Experiments were initially performed using Merck's Omnipure agarosepowder. Low yields of non-spherical particles ranging between 250 and300 μm were obtained. Experiments performed with an exaggerated amountof the hydrophilic emulsifier, 3.5 mL span 85 resulted in beads withoutpores but with a rough surface. By reducing the amount of thehydrophobic emulsifier, massive gellation due to the poor stabilizationof the agarose particles in the oil in water emulsion occurred. TABLE 1Effect of the stirring speed on the fabrication of porous agarose beadsAgarose aqueous solution concentration 4% (w/v), o/w emulsion: 0.7 mLtween 80/10 mL cyclohexane w/o emulsion: 7 mL span 85/75 mL cyclohexaneStirring speed with Fluorescence and Apparent Efficiency Size a magneticstirrer light microscopy porosity 250-300 μm 10 With oil A few Less thaninclusions, 10% regular integrity 9 Medium integrity None About 10% 8Better integrity A few but more About 10% than stir at 10

The effect of stirring speed has been briefly evaluated. With higherstirring speeds the integrity of the beads was poor. Smaller particlesare expected to be the result of faster stirring speeds, but exposure tohigher physical stress only results in the disintegration of the beads.Trials performed under the same conditions using Sigma agarose gavesimilar results to Merck agarose, but with slightly higher yields around20%. The integrity of the beads improved slightly suggesting bettermechanical properties such as gel strength.

Experiments for producing homogeneous particles were performed usingagarose obtained from Merck at a constant concentration of agarosesolution and stirring. The results are shown in Table 2. TABLE 2 Effectof the emulsifier on the fabrication of homogeneous agarose beadsAgarose aqueous solution concentration 4% (w/v), o/w emulsion: 0.7 mLtween 80/10 mL cyclohexane w/o emulsion: 7 mL span 85/75 mL cyclohexaneStirring speed with a Fluorescence and light Efficiency magnetic stirrermicroscopy Size 250-300 μm 10 Opaque beads About 10% 10 Regularintegrity About 10% 10 Bad integrity Less than 10%

Excessive stabilization of the water in oil emulsion causes reducedflocculation and increases the formation of fines resulting in a loweryield. Performing the same experiment with a fixed stirrer speed of 8(Corning stirrer/hot member, model # PC420) slightly increased theyield. Magnetic stirring may not be appropriate for viscous solutions orthe foam obtained during emulsification (creaming).

Bead Selection Techniques

Sensor arrays that use beads (either non-porous or porous) can be usedto determine the presence of a variety of analytes. Typically, the beadsinclude a receptor that binds to an analyte. The receptor may also bindto an indicator. The indicator typically produces a signal in thepresence of an analyte that is different from a signal produced in theabsence of an analyte. The selection of beads for use with a particularanalyte may be important to the success of the sensor array. In general,a bead should have a high affinity for the analyte and produce an easilydetectable signal. A method is described herein which may be used todetermine an optimal receptor for a given analyte and indicator.

One method used to determine the presence of an analyte is adisplacement assay. In a displacement assay a bead that includes areceptor is preloaded with an indicator. The indicator interacts (e.g.,is bound to) the receptor such that the bead appears to have a specificcolor or fluorescence due to the indicator. When a solution thatincludes an analyte is brought into contact with the bead, the analytemay displace the indicator from the receptor. This displacement maycause a loss of color or fluorescence of the bead since the indicator isno longer associated with the bead. By measuring the loss of color orfluorescence of the bead, the presence of an analyte may be determined.The success of such an assay for determining the presence of an analyteis dependent, in part, on the interaction of the receptor with theanalyte and the indicator. Generally, the bead should show a maximumcolor and fluorescence when an indicator is bound to the receptor,however, the indicator should be easily displaced by the analyte.

In one embodiment, a plurality of beads having a variety of receptorsmay be produced. In one embodiment, the receptors may be formed from avariety of different receptor types. Alternatively, the beads may havesimilar receptors. For example, techniques are well known to createlibraries of peptide, peptide mimics, or nucleotides upon polymericbeads. For peptide libraries up to 20^(n) different beads may beproduced in a library, where n is the number of amino acids in thepeptide chain. Nucleic acid libraries may have up to 4′ different beadswhere n is the number of nucleic acid bases. Because of the large numberof different beads in these libraries, the testing of each individualbead is very difficult.

FIG. 11 depicts a schematic drawing of a method for optimizing areceptor on a bead. In FIG. 11A, a bead is depicted that includes areceptor X. Receptor X is composed of 6 subparts that extend from abase. The base is coupled to the bead. The bead is first contacted withan indicator, denoted as the stars in FIG. 11A. The indicator interactswith each of the beads in the library, binding to the receptors. FIG.11B shows the indicator coupled to the receptor of the bead. As depictedin FIG. 11 b, the color or fluorescence of the bead is altered due tothe interaction of the indicator with the receptor. The change in coloror fluorescence of the bead indicates that the bead is capable ofinteracting with the indicator.

When a plurality of beads is used, the indicator will bind to the beadsat various strengths. The strength of binding is typically associatedwith the degree of color or fluorescence produced by the bead. A beadthat exhibits a strong color or fluorescence in the presence of theindicator has a receptor that binds with the indicator. A bead thatexhibits a weak or no color or fluorescence has a receptor that onlyweakly binds the indicator. Ideally, the beads that have the bestbinding with the indicator should be selected for use over beads thathave weak or no binding with the indicator. FIG. 12 depicts a schematicof a flow cytometer that may be used to separate beads based on theintensity of color or fluorescence of the bead. Generally, a flowcytometer allows analysis of each individual bead. The beads may bepassed through a flow cell that allows the intensity of color orfluorescence of the bead to be measured. Depending on the measuredintensity, the bead may be collected or sent to a waste collectionvessel, as indicated in FIG. 12. For the determination of an optimalbead for interaction with an indicator, the flow cytometer may be set upto accept only beads having an color or fluorescence above a certainthreshold. Beads that do not meet the selected threshold, (i.e., beadsthat have weak or no binding with the indicator) are not collected andremoved from the screening process. Flow cytometers are commerciallyavailable from a number of sources.

After the bead library has been optimized for the indicator, the beadsthat have been collected represent a reduced population of theoriginally produced beads. If the population of beads is too large,additional screening may be done by raising the intensity threshold. Nowthat the beads that exhibit optimal interaction with a receptor havebeen identified, the remaining beads are optimized for displacement ofthe indicator by the analyte of interest. Thus, the remaining beads aretreated with a fluid that includes the analyte of interested, asdepicted in FIG. 11C. The analyte is represented by the circle. For somebeads, the analyte will cause displacement of the indicator, causing thecolor or fluorescence of the bead to be reduced, as depicted in FIG. 1ID. The intensity of the color or fluorescence of the bead after itinteracts with an analyte will be based on how the competitivedisplacement of the indicator. A bead that exhibits weak or no color orfluorescence when treated with an analyte is the most desirable. Suchbeads show that the analyte is readily bound by the receptor and canreadily displace the indicator from the receptor.

Once again a flow cytometer may be used to determine the optimal beadsfor use in an assay. A library of beads that have been optimized forinteraction with an indicator are treated with a fluid that includes ananalyte. The treated beads are passed through a flow cytometer and thebeads are separated based on intensity of color or fluorescence. Thebeads that exhibit a color or fluorescence below a predeterminedintensity are collected, while beads that show a color or fluorescenceabove the predetermined intensity are sent to a waste collection. Thecollected beads represent the optimal beads for use with the selectedanalyte and indicator. The identity of the receptor coupled to the beadmay be determined using known techniques. After the receptor isidentified, the bead may be reproduced and used for analysis of samples.

The previously described sensor array systems and membrane-based systemsmay be used in diagnostic testing. Examples of diagnostic testing aredescribed in U.S. application Ser. No. 10/072,800, entitled “METHOD ANDAPPARATUS FOR THE CONFINEMENT OF MATERIALS IN A MICROMACHINED CHEMICALSENSOR ARRAY” filed Jan. 31, 2002 and published as U.S. Publication No.2002-0197622-A1.

In many common diagnostic tests, antibodies may be used to generate anantigen specific response. Generally, the antibodies may be produced byinjecting an antigen into an animal (e.g., a mouse, chicken, rabbit, orgoat) and allowing the animal to have an immune response to the antigen.Once an animal has begun producing antibodies to the antigen, theantibodies may be removed from the animal's bodily fluids, typically ananimal's blood (the serum or plasma) or from the animal's milk.Techniques for producing an immune response to antigens in animals arewell known.

Once removed from the animal, the antibody may be coupled to a polymericparticle. The antibody may then act as a receptor for the antigen thatwas introduced into the animal. In this way, a variety of chemicallyspecific receptors may be produced and used for the formation of achemically sensitive particle. Once coupled to a particle, a number ofwell-known techniques may be used for the determination of the presenceof the antigen in a fluid sample. These techniques includeradioimmunoassay (RIA), microparticle capture enzyme immunoassay (MEIA),fluorescence polarization immunoassay (FPIA), and enzyme immunoassayssuch as enzyme-linked immunosorbent assay (ELISA). Immunoassay tests, asused herein, are tests that involve the coupling of an antibody to apolymeric particle for the detection of an analyte.

ELISA, FPIA and MEIA tests may typically involve the adsorption of anantibody onto a solid support. The antigen may be introduced and allowedto interact with the antibody. After the interaction is completed, achromogenic signal generating process may be performed which creates anoptically detectable signal if the antigen is present. Alternatively,the antigen may be bound to a solid support and a signal is generated ifthe antibody is present. Immunoassay techniques have been previouslydescribed, and are also described in the following U.S. Pat. Nos:3,843,696; 3,876,504; 3,709,868; 3,856,469; 4,902,630; 4,567,149 and5,681,754.

In ELISA testing, an antibody may be adsorbed onto a polymeric particle.The antigen may be introduced to the assay and allowed to interact withan antibody for a period of hours or days. After the interaction iscomplete, the assay may be treated with a dye or stain, which reactswith the antibody. The excess dye may be removed through washing andtransferring of material. The detection limit and range for this assaymay be dependent on the technique of the operator.

Microparticle capture enzyme immunoassay (MEIA) may be used for thedetection of high molecular mass and low concentration analytes. TheMEIA system is based on increased reaction rate brought about with theuse of very small particles (e.g., 0.47 μm in diameter) as the solidphase. Efficient separation of bound from unbound material may becaptured by microparticles in a glass-fiber matrix. Detection limitsusing this type of assay are typically 50 ng/mL.

Fluorescence polarization immunoassay (FPIA) may be used for thedetection of low-molecular mass analytes, such as therapeutic drugs andhormones. In FPIA, the drug molecules from a patient serum and drugtracer molecules, labeled with fluorescein, compete for the limitedbinding sites of antibody molecules. With low patient drugconcentration, the greater number of binding sites may be occupied bythe tracer molecules. The reverse situation may apply for high patientdrug concentration. The extent of this binding may be measured byfluorescence polarization, governed by the dipolarity and fluorescentcapacity.

Cardiovascular risk factors may be predicted through the identificationof many different plasma-based factors using immunoassay. In oneembodiment, a sensor array may include one or more particles thatproduce a detectable signal in the presence of a cardiac risk factor. Insome embodiments, all of the particles in a sensor array may producedetectable signals in the presence of one or more cardiac risk factors.Particles disposed in a sensor array may use an immunoassay test todetermine the presence of cardiovascular risk factors. Further detailsregarding the use pf particle based sensor arrays for the detection ofcardiac risk factors may be found in U.S. patent application Ser. No.10/427,744 entitled “Method and System for the Detection of Cardiac RiskFactors” (Published as U.S. Publication No.: 2004-0029259-A1) and U.S.patent application entitled “Method and System for the Analysis ofSaliva Using a Sensor Array” to McDevitt et al., filed on Dec. 13, 2004.

The sensor array may be adapted for use with blood. Other body fluidssuch as, saliva, sweat, mucus, semen, urine and milk may also beanalyzed using a sensor array. The analysis of most bodily fluids,typically, will require filtration of the material prior to analysis.For example, cellular material and proteins may need to be removed fromthe bodily fluids. As previously described, the incorporation of filtersonto the sensor array platform, may allow the use of a sensor array withblood samples. These filters may also work in a similar manner withother bodily fluids, especially urine. Alternatively, a filter may beattached to a sample input port of the sensor array system, allowing thefiltration to take place as the sample is introduced into the sensorarray.

In an embodiment of a sensor array, particles may be selectivelyarranged in micromachined cavities localized on silicon wafers. Thecavities may be created with an anisotropic etching process as describedin U.S. application Ser. No. 10/072,800, entitled “METHOD AND APPARATUSFOR THE CONFINEMENT OF MATERIALS IN A MICROMACHINED CHEMICAL SENSORARRAY” filed Jan. 31, 2002 and published as U.S. Publication No.2002-0197622-A1.

In some embodiments, to observe the sensor array, a flow cell is mountedupon the stage of an optical imaging system To accommodate variousdetection schemes, the imaging system is outfitted for both brightfieldand epifluorescence imaging. Appended to the imaging system is acomputer controlled CCD camera, which yields digital photomicrographs ofthe array in real time. Use of a CCD may allow multiple optical signalsat spatially separated locations to be observed simultaneously.Digitization also permits quantification of optical changes, which isperformed with imaging software. As mentioned earlier, the flow cell isreadily compatible with a variety of fluidic accessories. Typically,solutions are delivered to the flow cell with the assistance of a pump,often accompanied by one or more valves for stream selection, sampleinjection, etc.

As fluid samples are delivered to the flow cell, optical responses ofthe sensor array are observed and reported by the CCD camera. As such,the raw data produced by this platform are digital opticalphotomicrographs. Once an image has been captured, quantification of theparticles' responses begins. Multiple areas of interest (AOIs) aredefined within each image, typically corresponding to the individualparticles. Average red, green, and blue (R, G, and B, respectively)pixel intensities are determined for each AOI, and exported as the rawnumerical data. Software modules have been composed allowing many ofthese tasks to be performed in an automated fashion. Automated tasksinclude periodic acquisition of images, determination of AOIs(recognition of particles), extraction and exportation of numerical datato spreadsheet, and some data manipulation.

Several manipulations of the RGB intensities may be quantified for eachparticle in the array. In addition to the indicator particles, blankparticles (ones containing no receptors or indicators) were alsoincluded in the array to serve as references for absorbancemeasurements. The R_(n), G_(n), and B_(n) values were used to refer tothe average intensities, in each color channel, for particle n.Similarly, R₀, G₀, B₀ values represented the average intensities, ineach color channel, for a blank reference particle. “Effectiveabsorbance” values for each color channel, A_(Rn), A_(Gn), and A_(Bn),were then calculated using equations 3.1-3.3.A _(Rb)=−log(R _(n) /R ₀)  Eq. 3.1A _(Gn)=−log(G _(n) /G ₀)  Eq. 3.2A _(Bn)=−log(B _(n) /B ₀)  Eq 3.3

These effective absorbance values were also normalized to their maximumvalue for a given experiment and were referred to as A′_(Rn), A′_(Gn),A′_(Bn). The ratios of a given particle's different color intensitiesmay also be calculated. For a given particle, n, the ratio of the redintensity over the green intensity was expressed as (R:G)_(n), that ofred over blue as (R:B)_(n), and that of green over blue as (G:B)_(n).

In order to create an array with broad analyte response properties andaccurate measurement capabilities, it is necessary to develop proceduresfor translating optical changes into analyte quantification values.Here, the collective response of numerous particles and selective colorchannels must be considered. For this purpose, artificial neural network(ANN) methods were utilized due to their capacity to process multipleinputs. Multilayer Feedforward ANNs are the most popular ANNs and arecharacterized by a layered architecture, each layer comprising a numberof processing units or neurons. An explanation of how a multi-layer ANNfunctions is facilitated by the schematic diagram provided in FIGS. 13Aand B. In FIG. 13A is shown a generic representation of a multi-layerANN. There is both an input layer and an output layer. The number ofneurons in the input layer is typically equal to the number of datapoints to be submitted to the network. On the other hand, the number ofneurons in the output layer may vary with the nature of the application(e.g. either one or multiple values may be appropriate as the network'soutput). Layers between the input and output are termed “intermediate”or “hidden” layers. Inclusion of hidden layers greatly increases anetwork's capabilities. However, there is a concomitant increase incomplexity, which rapidly becomes computationally cumbersome, even withmodern computers. Likewise, it is desirable to identify ANN methods thatare both simple, yet effective, for the given application goals.

When data are submitted to the input layer of such an ANN, correspondingresults are yielded in the output layer. The transformation of the datainto the results occurs as the data or “signal” progresses through thelayers of the network. To reveal how these transformations are made,FIG. 13B focuses on the interactions between three layers in amulti-layer ANN. From each neuron (1, 2, . . . , n) in the precedinglayer, the centrally featured neuron receives an individual input (in₁,in₂, . . . , in_(n)). The neuron has a number of weight values (w₁, w₂,. . . , w_(n)) which correspond to the received inputs. The neuronassigns a weight to each of these inputs and subsequently calculatestheir weighted sum, S: $\begin{matrix}{S = {\sum\limits_{n}^{1}{{in}_{n}*w_{n}}}} & {{Eq}.\quad 3.4}\end{matrix}$An output (out) is then generated by passing this weighted sum of inputsthrough a sigmoidal function,out=f(S)=1/(1+exp−S)  Eq. 3.5effectively narrowing the potential output range. This output value isthen sent to every neuron in the subsequent layer of the network.Connecting lines between the neurons (such as those in FIG. 13A) aretypically used to demonstrate that each neuron has such interactionswith every neuron in the layers immediately preceding and following itsown.

The accuracy (and consequent utility) of an ANN may be dependent uponits training. The training methods that may be utilized may be eitherthe Levenberg-Marquardt (LM) algorithm or the Back Propagation algorithm(BP). The BP algorithm Typically, training involves gathering a large,representative data set (e.g., a simple calibration curve) anddesignating it as a training data set, including both inputs andcorresponding desired outputs. Both the inputs and the desired outputsare supplied to the network, which then refines itself in an iterativemanner. The network (whose architecture has been chosen by the user)processes the supplied inputs, yielding a set of outputs. These outputsare generated in the manner described above, initially using randomvalues for the neurons' weights. The use of random weights producesnonsensical results, but provides the network with a necessary startingpoint. The network then refines itself by comparing its produced outputswith the desired outputs, and then altering its neurons' weights for thesubsequent iteration in order to decrease the difference between thetwo. Each cycle comprising input submission, output generation, andweight adjustments, is referred to as an epoch. Training proceeds for auser-defined number of epochs, often on the order of 1000, even forrelatively simple networks.

Once an ANN has been trained, the difference between the desired outputsof the training data set and the outputs actually generated by thenetwork is quantified as the training error. Obviously, minimal trainingerrors are desired. High training errors may be due to any number offactors, but can often be attributed to network architecture orinsufficient training. More complex architecture (i.e., more layersand/or more neurons per layer) may improve the training error, but mayalso greatly increase the time and computational power required fortraining and use.

To assess the predictive ability of an ANN during the training process,a second iterative process may be employed. In a given iteration of thisprocess, a single data point from the training data set is omitted, theANN is trained on the remaining data, and then tested on the omittedpoint. This “leave-one-out” strategy is useful for evaluating thenetwork's ability to extrapolate. It should be kept in mind, though,that this is a pseudo-extrapolation (in that the omitted test pointoriginated in the training data). As such, the average error associatedwith this pseudo-external data is typically lower than that of trulyexternal data (data gathered outside of the original training data set).The error measured when the ANN is used on truly external data is themost meaningful measure of the network's utility. However, many reportsof chemical sensor arrays employing ANNs fail to distinguish betweenerror values associated with truly external data and pseudo-externaldata. The extraction of intuitively useful trends is often difficultfrom many ANN studies described in the literature, making the targetedimprovement of array members difficult.

Values of R_(n), G_(n), B_(n); A_(Rn), A_(Gn), A_(Bn), and (R:G)_(n),(R:B)_(n), (G:B)_(n), are all considered for participation in thetraining network as input data. Raw intensity inputs such as R_(n),G_(n), B_(n) are discarded early on in this study because they are foundto be highly dependent on the light calibration setting and the size ofthe particle. However, using a “blank” particle to convert rawintensities to “effective absorbance” results in measurements that takeinto account possible fluctuations of the light source during the courseof an experiment. As mentioned above, ANNs may be sensitive to theformat of the inputs and sometimes necessitate the completion of datatransformation or pre-processing of the inputs. Normalization of theabsorbance readings homogenizes the data by transforming everymeasurement into a value between 0 and 1. Therefore, “effectiveabsorbance” readings are also discarded as inputs in the network andreplaced by A′_(Rn), A′_(Gn), A′_(Bn). This switch presumably reducesthe influence of error caused by variations in particle diameter. Theuse of color ratios provides a second method to reduce the noisecontribution introduced by the selection of particles with a slightdistribution in their sizes.

For network training, evaluation, and method selection, every recordeddata set may contain replicates (or cases) for each data point throughthe acquisition of a sequence of images. Preliminary experiments testedthe influence of the number of cases on the accuracy of the network. Themain advantage of using multiple cases is to provide complex networkswith a much greater number of data points than the number of connectionsbetween neurons. Further, the procedure allows for some of the data tobe used in cross-validation. It is generally recommended that the numberof training cases be at least twice that of adjustable parameters in thenetwork. The number of epochs necessary to train a given network may beassessed carefully by first introducing cross-validation cases in thetraining set. The inclusion of cross-validation data does not enhancethe performance of the network to any great extent, but rather serves tolimit the number of over-fitting occurrences. All data collection eventsare completed with at least one duplicate of each particle, and the samefor the blank particle. The use of redundant inputs is intended to notonly provide a back-up for each data type, but also to serve to increasethe dimensionality of the network in order to optimize patternrecognition. However, despite the good particle-to-particlereproducibility observed in prior experiments, the performance of thenetwork is found consistently to be greater with a single replicate foreach particle rather than taking average values recorded from multiplesimilar type particles.

The preparation of functional shells within the polymer microspheres wasaccomplished via methods based on those outlined by Fourkas andcoworkers (Farrer, R. A. et al. “Production, analysis, and applicationof spatially resolved shells in solid-phase polymer spheres”, Journal ofthe American Chemical Society 124, 1994-2003 (2002)). Syntheticmodification of a given microsphere entails immobilization of a speciesto the reactive sites of the particle. Intuitively, this begins at theparticle's surface and proceeds inward in a radial manner. In the eventthat the coupling reaction between the solution borne species and theparticle's reactive sites occurs more rapidly than the species'diffusion into the particle, the advancing reaction front will remainabrupt. At any point during the reaction, then, there are two distinctregions: a growing exterior region in which the reactive sites have beenmodified and a shrinking, unmodified core region. Thus, if the reactionis aborted prior to completion (i.e., before the advancing reactionfront reaches the center of the particle) it will yield a microspherewith two distinct concentric regions. In theory, multiple suchcontrolled-penetration reactions can be performed sequentially to yieldadditional shells.

As mentioned above, the utility of this technique is limited toscenarios in which diffusion of the species to be immobilized is therate limiting step. If this is not the case, definition of the regionsmay be very poor or even nonexistent. Recently, however, Farrer et alreported an indirect method for the creation of discrete regions withinpolymer microspheres which circumvents the issue of diffusion vs.reaction rates, vastly broadening the range of species which may beimmobilized in distinctly defined shells. Instead of directlyimmobilizing the desired species, temporary shells were created bycapping peripheral reactive sites with a removable protecting group.With an exterior protected shell in place, the internal core region ofthe particle may be modified with a subsequent coupling reaction.Removal of the protecting group from the external region then yields aparticle in which the core has been modified, but the exterior has not.In this manner, multishell particles are prepared from the core outward.Again, repeated protection/modification/deprotection cycles may beperformed sequentially to increase the number of shells.

The key advantage to this indirect modification technique is that thesharpness of the interface between two shells is established by theprotecting group. Variations on this technique, including the generationof five or more layers within individual particles, the simultaneous useof multiple orthogonal protecting groups, and the spatially resolvedimmobilization of three different species within particles. In all ofthese variations, though, the controlled penetration of the protectinggroup is used to define the shells. Thus, the spatial resolution of theshells is independent of the diffusion and reaction rates of the speciesto be immobilized within them.

FIG. 14 displays schematically the synthesis of functional multi-shellparticles. Initially, distinctly heterogeneous regions are createdwithin the amine terminated polystyrene-polyethylene glycol particles(i) via the controlled penetration of the resin in a radial manner with9-fluorenylmethoxycarbonyl chloroformate (Fmoc), yielding resin with anexterior region of protected amines (ii). Subsequent coupling of ALZC toii results in particles with the complexone immobilized only withintheir cores (iii). Removal of the Fmoc protecting group then yieldsresin with an ALZC core and an exterior region of free amines (iv). Twoaliquots of iv are individually treated with acetic anhydride and EDTAdianhydride, respectively, yielding two batches with identical cores,but different exterior regions. While batch vi is functionalized with astrongly chelating EDTA shell, the amines in the exterior of batch v arecapped, rendering the shell relatively inert with respect to metalcations. Multishell particle types will be named by combining theirfunctionalities, listing them from the exterior inwards. For example,particles from batch vi in FIG. 14 will be referred to as “EDTA-ALZC”particles.

Particles from batches v (Ac-ALZC) and vi (EDTA-ALZC) were arranged in asensor array with each truncated pyramidal well hosting an individualparticle, directing solution flow to the particle while allowing opticalmeasurements to be made. The red, green, and blue absorbance values(calculated using a blank particle as a reference intensity, aspreviously described) of each particle were monitored vs. time asvarious metal cation solutions were delivered to the flow cell. In oneexperiment, RGB absorbance was measured vs. time for a particle frombatch v and a particle from batch vi, during a representative experiment(specifically the introduction of 10 mM Ni²⁺). Both particles exhibit anoverall increase in absorbance, as was expected from the ALZC “detector”core. In the particle with the “inert” acetylated shell, (A,C) theabsorbance increase begins roughly 8 s after the Ni²⁺ flow begins. Thisvalue was constant from particle to particle (within Batch v) and alsofrom trial to trial. In contrast, the absorbance increase was notobserved in the EDTA-coated particles (Batch vi) until ˜40 s later. Thisdelay is consistent with the idea that the ligand shell hinders thediffusion of metal cations through the polymer matrix.

It is also interesting to note that the two different particles havevery different absorbance values prior to arrival of the metal cationsolution. Here, it is speculated that ligand groups in the outer shellsmay function to buffer the microenvironments of the particles, therebyplaying a role in dictating the color of the detection scheme. Withhigher concentration acidic and basic rinses, the color of the ALZC inthe two batches of particles was readily equalized. However, with the 50mM acetate buffer used here, the different particle batches consistentlyexhibited different (but stable) absorbance values, as consistent withthe above explanation. Further, it should be noted that for the EDTAparticle (batch vi, panels B and D) a decrease in absorbance wasobserved prior to the overall increase in absorbance. This behavior isconsistent with a temporary lowering of the pH of the particlemicroenvironment, which may be attributed to deprotonation of theligands upon metal complexation, and has been observed in relatedsystems. Recent data indicate that this feature of the multishellparticles' responses may be useful in identifying metals and determiningtheir concentrations.

The delayed response of the EDTA coated particle can be rationalized interms of a “moving boundary” or “shrinking core” effect. The diagram inFIG. 15 illustrates the shrinking-core model as it pertains to amicrosphere functionalized homogeneously with a chelating moiety (i.e.,iminodiacetate resin). The lower portion of the figure contains a pairof graphs, one depicting the concentration of metal in solution as afunction of radial position within the particle, the other displayingthe concentration of metal bound by the solid resin, also as a functionof radial position. The two graphs are oriented in opposing directions(separated by a dashed line) such that the radial positions on thex-axis of each correspond to the semicircular diagram of a microsphere,included above them.

Upon exposure to solution containing an analyte (e.g., metal cations),the concentration gradient between the interior of the particle and thesurrounding solution prompts diffusion of the analytes into theparticle. However, given a large formation constant between the ligandand the analyte, the analytes achieving contact with the polymer may beassociated (e.g. through binding or complexation) with the polymer,removing solution dissolved analytes from the liquid. This effectiveconsumption of the analytes as they progress through the polymer resultsin the preservation of a large concentration gradient across awell-defined, moving boundary. Consequently, at a given point in timeprior to complete equilibration, there are two distinct regions in themicrosphere: a reacted shell and an unreacted core, as shown in FIG. 15.The shell is defined by local equilibrium between the solution and thepolymer matrix. Accordingly, the two concentration profiles shown in theschematic suggest the presence of both free and bound analytes in thisregion. If equilibration is achieved rapidly, the concentrations of eachwould be expected to remain approximately constant throughout the shell.The core, on the other hand, is defined by an absence of any analytes,neither free nor bound forms are here located at this time interval. Assuch, there exists a concentration gradient across the boundary(indicated with dotted lines) between the two regions. Thisconcentration gradient naturally promotes mass transport of the analytesacross the boundary. However, since the interaction of the analytes withthe polymer occurs more rapidly than their diffusion, the net result isan inward shift of the boundary with the concentration gradientpreserved. It should be noted that the existence of the two regions istransient, and that, with prolonged time intervals, the entire particlewill attain equilibrium with the analyte resulting in a homogeneoussystem.

In the EDTA-ALZC particle described above here, arrival of the boundaryat the dye-containing core is signaled by the increase in absorbance.Following the initial arrival at the core, there continues to be aslower rate of signal development compared to the reference Ac-ALZCparticle. This behavior may be indicative of the fact that theconcentration gradient is not perfectly maintained, or rather, that theboundary region broadens as it progresses through the matrix. Also, itshould be kept in mind that the EDTA-ALZC particle used here differssomewhat from the homogeneous particle discussed in the model. Inparticular, we must consider that the ALZC core is also an immobilizedchelator, and as such that the rate of signal development will also bedependent upon interactions between the metal and the dye. Furthermore,if complexation of metal ions by the ligand shell does indeed affect thepH of the particle microenvironment, as proposed above, it may alsosignificantly affect the binding characteristics of the complexometricdye. Nevertheless, the model provides a qualitative explanation of thekey processes that may occur within the particle as metal cations areincorporated therein.

In order to facilitate an examination of the benefits of this multishellapproach, three key intuitive components of a particle's response aredefined as follows: 1) the color change of a particle is calculated bysubtracting its initial effective absorbance value from its finaleffective absorbance value; 2) t_(D) is the time measured from thebeginning of a particle's color change until the particle has completedhalf of its color change; 3) t_(L) is the time required to penetrate theligand shell as defined by the length of time prior to the observationof the color change. These components of the particles' responses can becombined to yield a multi-component “fingerprint” summarizing thearray's response to a given metal cation solution.

Examples of such multi-component responses are graphically summarized inFIGS. 16A-D for the particles prepared according to the scheme of FIG.14. Each of the four panels here included corresponds to the indicatedmetal solution and features two separate data sets associated with EDTAand acetylated outer shells. Interestingly, the fingerprints yielded bythe two multishell particles exhibit unique characteristics for each ofthe solutions studied. These data are well-suited for use with patternrecognition algorithms. A comparison of FIG. 16C (5 mM Pb²⁺) and FIG.16D (10 mM Pb²⁺) emphasizes the benefits of the increased dimensionalityof the fingerprint response. While the color changes exhibited by thetwo particle types show little, if any, meaningful difference betweenthe two concentrations, the t_(D) values of both particles, and thet_(L) values of the EDTA particle, differ significantly between the twoconcentrations. It is evident from these data that the final staticcolorimetric response (the color change) of the ALZC alone isinsufficient for discriminating between the two concentrations of Pb²⁺,and that the functional EDTA shells and the time domain have added tothe array's capabilities. Conversely, in the cases displayed in FIG. 16A(10 mM Zn²⁺) and FIG. 16B (10 mM Ni²⁺) the t_(D) and t_(L) values of theparticles differ only slightly between the two metals, while their colorchanges are distinctly different. For these cases, the calorimetricresponses of the ALZC contribute more to the discrimination than do thetemporal components of the response. Likewise, a comparison of panel D(10 mM Pb²⁺) with either panel A (10 mM Zn²⁺) or B (10 mM Ni²⁺)demonstrates a situation in which both the temporal and calorimetriccomponents differ between metals. That the t_(L) values of theacetylated (v) particle do not fluctuate significantly between thesefour cases agrees well with the idea of an “inert” shell, and highlightsthe chromatographic role provided by the EDTA functionality.

It is important to appreciate that with the multishell approach usedhere, the polymer microsphere itself is the sensor element, rather thanmerely a substrate for immobilization of a detection scheme. Whileoptical detection of the analytes still arises from the immobilizedindicator, modification of the polymer matrix surrounding the indicatormay be used to augment the analytical characteristics of the detectionscheme. Consequently, preparing particles with different ligand shells,but having a common indicator core generates a collection ofcomplementary sensing elements with overlapping selectivity and variedanalytical characteristics. Such elements are the building blocks ofcross-reactive sensor arrays. It should be emphasized here that this isaccomplished without any direct synthetic modification of the indicatoritself.

In order to investigate the advantages of varying the nature of theligand shell, a new batch of multishell particles was prepared.Preparation followed the strategy outlined previously and is depictedschematically in FIG. 17. As before, the controlled penetration of Fmocwas employed to generate a batch of NH₂-ALZC resin. Four aliquots ofthis resin were removed and the exterior regions of each aliquot wasmodified independently. In addition to capping the amines in one aliquotvia acetylation, and immobilizing EDTA in the shell of a second, twoother polyaminocarboxylate ligands, nitrilotriacetic acid (NTA) anddiethylenetriaminepentaacetic acid (DTPA), were immobilized in theshells of the remaining two aliquots. The DTPA ligand system wasimmobilized in a similar fashion as EDTA, via DTPA dianhydride, where asNTA was immobilized similarly to the complexometric dye, via a DCCcoupling reaction.

Samples of the four particle types prepared here were assembled in asensor array in order to probe the effects of the different ligands onthe particles' responses. The “split-pool” preparation of theseparticles (described above) ensures that the shell depth and dye coreare identical (within the tolerances described in later) from batch tobatch. Accordingly, any observed significant differences in t_(L) valuesbetween batches may be attributed to their respective ligands, ratherthan differences in shell depth. Different concentration solutions ofCa(NO₃)₂ and Mg(NO₃)₂ were introduced to the array and plots ofabsorbance vs. time were generated for each particle in the array.Solutions contained only a single metal (i.e., either Ca²⁺ or Mg²⁺) andtheir concentrations ranged from 5 μM to 10 mM. All solutions werebuffered at pH 9.8 with 50 mM alanine. The duration of each trial variedwith the anticipated t_(L) values. One image was captured every 2 s.

FIG. 18 features plots of the t_(L) values of three different particletypes (NTA-ALZC, EDTA-ALZC, and DTPA-ALZC) vs. metal concentration forboth Mg²⁺ and Ca²⁺. An examination of these data reveals severaladvantages of the multi-shell approach. It is evident from the data thatall three ligand shells employed here exhibit dose dependent responsesfor both Ca²⁺ (empty circles, dashed lines) and Mg²⁺ (filled circles,solid lines). This concentration dependence of the t_(L) valuesindicates that the ligand shells should be directly applicable toconcentration determination. Furthermore, it should be noted that for agiven metal the dose dependence of each ligand shell shown here issignificantly different. This agrees well with the intuitive notion thatthe t_(L) value should be heavily dependent upon the identity of theligand in the exterior region. This then implies that the t_(L) value ofeach ligand shell should be useful over a different range of metalcation concentration. If this is indeed the case, then by combiningparticles with various ligand shells, it should be possible to extendthe effective dynamic range of an array towards a given metal cation.Additionally, although the EDTA and DTPA shells appear to treat Ca²⁺ andMg²⁺ very similarly, the NTA shells clearly discriminate between the twometals. As such, the NTA ligand shell can be considered to impart adegree of selectivity to a particle.

In an experiment, multiple samples of a 10 mM Pb²⁺ solution (buffered atpH 4.8 with 50 mM alanine) were delivered to an array of multishellparticles, and their responses were recorded. The 5×7 array used in thiswork contained 7 of each of the 5 following particle types: blank (NH₂),Ac-ALZC, NTA-ALZC, EDTA-ALZC, and DTPA-ALZC. Between each trial, anacidic rinse (10 mM HCl at 3 mL/min for ˜15 min) was used in an attemptto remove bound Pb²⁺ from the particle. The acidic rinse was followed bya buffer rinse (2 mL/min for ˜5-7 min) to ensure a uniform startingpoint for each trial. Images of the array were captured every twoseconds and an absorbance vs. time plot was recorded for each particlein the array. From these responses, a t_(L) value was extracted for eachparticle, for each trial. For a given particle, the t_(L) value wasquantified by taking the slope of the slope of the particle's greenabsorbance vs. time and observing the peak which corresponded to themost rapid rate of increase in absorbance. In each case, this methodyielded values which agreed well with visual inspections of the rawdata.

Mean t_(L) values were calculated for individual particles by averagingt_(L) values from the five redundant trials.

Several observations were made concerning the particles' temporalreproducibility. First, different ligand shells exhibited differentt_(L) values for the 10 mM Pb²⁺ solution. This suggests that theinclusion of multiple ligand types should contribute to the generationof fingerprint style responses. Additionally, the average standarddeviations for the different particle types are as follows: 1.3 s forAc-ALZC; 2.6 s for NTA-ALZC; 1.6 s for EDTA-ALZC; 3.5 s for DTPA-ALZC.Considering that the temporal resolution of the measurements was only 2s, and that the reproducibility was also dependent upon manualsynchronization of two independent software packages (one controllingfluid delivery, one controlling image capture), these data are veryencouraging with respect to trial-to-trial reproducibility. Furthermore,since the time of these studies, it has been observed that the acidicrinse used here is inadequate for the DTPA ligand shell. This may wellhave contributed to the modest reproducibility exhibited here by theDTPA coated particles.

Concerning particle-to-particle reproducibility, the absolute andpercent relative standard deviations (% RSD) of the average t_(L) valuesfor each particle type are as follows: 1.1 s, 9.3% for Ac-ALZC; 13.8 s,13.9% for NTA-ALZC; 1.6 s, 4.9% for EDTA-ALZC; 3.4 s, 7.8% forDTPA-ALZC. It is encouraging that, in this initial study, only theNTA-ALZC particles' responses exhibited % RSDs greater than that of theshell depth (9.9%). It is possible that uneven solution flow through thewells of the array results in unequal delivery of analyte and thereforehampers particle-to-particle reproducibility. If this is indeed thecase, it would not be surprising if it was most evident in the particleswith the highest t_(L) values.

The ligand shell of a multishell particle can be thought of as achromatographic layer, while the indicator at the core functions as adetector. Indeed, data presented thus far have indicated that theprogression of analytes through the particles' exterior regions ishindered by the presence of an immobilized ligand and that the rate ofprogression is dependent upon the nature of the ligand and the identityand concentration of the analyte. Certainly, in their interactions withindividually delivered analytes, the multishell particles havedemonstrated a potential utility for metal cation speciation andconcentration determination. It should be kept in mind though that theprimary goal of cross-reactive sensor arrays is the ability to detectmultiple species simultaneously.

The plot displayed in FIG. 19 chronicles the development of an EDTA-ALZCparticle's response to a solution containing both Mg²⁺ and Ca²⁺. The topline represents the green absorbance, the middle line represents the redabsorbance, and the top line represents the blue absorbance. Each metalwas present at a concentration of 1 mM, the solution was buffered at pH9.8 with 50 mM alanine, and the flow rate during the experiment was 2mL/min. As was seen with the introduction of single cations, there is asignificant delay prior to observation of the dye's response. However,the evolution of the dye's response is clearly different here than withany of the individually delivered analytes. Specifically, the observedcolor change appears to occur in two distinct steps, the firstcommencing roughly 115 s after the beginning of sample introduction, thesecond beginning almost 100 s later. This is most readily evident in theresponse recorded by the red channel (middle line) of the CCD. Thepresence of these two steps, and the plateau between them, is indicativeof two samples arriving at the dye core of the particle at differenttimes, suggesting that the EDTA shell may have actually separated thetwo species during their progression through the exterior region. Itshould also be noted that the two steps in the signal development differspectrally. The first step is defined by an absorbance increase whichspans all three channels of the CCD, whereas the second step is observedprimarily in the red channel, slightly in the green channel, and not atall in the blue. This bathochromic shift in the dye's absorbance agreeswith the idea of two cation waves of different composition arriving atthe dye core at different times.

Interpretation of the microsphere's response is again facilitated by aconsideration of a moving boundary scenario. In FIG. 20 a diagram isused to illustrate the model developed by Mijangos and Diaz for a movingboundary system involving two species of metal cations. The arrangementand format of the diagram match that of FIG. 15. For this example, thesame concentration of each species has been introduced to themicrosphere, and the ligating polymer matrix is assumed to bind eachspecies with a different affinity. Additionally, the diffusivities ofthe two species are taken to be identical. On each graph theconcentrations (free or bound as indicated on the y-axes) of the twocations are shown. The dashed plots (- - - -) correspond to the analytewith the higher affinity for the matrix, the solid plots correspond tothe less preferred analyte.

Upon sample introduction, both analytes are subject to a concentrationgradient between the external solution and the particle. Consequently,both diffuse into an outer shell of the particle in equal concentrationswhere they are bound differentially by the immobilized chelator. Thispreferential binding establishes a different concentration gradient foreach species. The solution in the shell has been depleted of the higheraffinity species, and so its gradient effectively remains at the surfaceof the particle. On the other hand, the less preferred analyte is stillpresent in solution in relatively high concentrations and so itexperiences a gradient between the outer shell and the inner region.Diffusion of the two species in accordance with the described gradientsresults (temporarily) in a situation similar to that depicted in FIG.20.

The two concentration gradients in solution (depicted in the left handgraph) explain both the encroachment of region 2 on the unreacted core,and that of region 1 on region 2. Region 2 contains only the lesspreferred analyte and progresses into the core as in the monoanalytesystem described previously. In contrast, the outer region (1) containsboth species, and its progression (also driven by a concentrationgradient in solution) entails the displacement of the less preferredanalyte from the chelating matrix.

According to the model described above, the two steps within theEDTA-ALZC particle's response should correspond to the arrival of asingle analyte at the dye core followed by the arrival of a mixture ofthe two analytes. The time dependent 3-color absorbance curves providedin FIGS. 21A-C allow us to begin rationalizing the features seen withinthe bianalyte response. In FIG. 21A-C, the top line represents the greenabsorbance, the middle line represents the red absorbance, and the topline represents the blue absorbance. These plots show three differentresponses from an EDTA-ALZC particle. FIGS. 21A and 21B show theparticle's response to 2 mM Ca(NO₃)₂ and 2 mM Mg(NO₃)₂, respectively.Each response exhibits a delay, as expected, and each response isspectrally different also. While the dye's response to Mg²⁺ appearssimply to be an increase in absorbance, the Ca²⁺ solution elicits notonly an increase in absorbance, but also a significant spectral shiftinto the red channel of the CCD. These two monometallic responses aid ininterpretation of the bimetallic response shown in FIG. 19, implying thepresence of Ca²⁺ in the second step of the signal development, and itsabsence from the first.

FIG. 21C shows an EDTA-ALZC particle's response to the sequentialdelivery of two different samples, the first consisting of 5 mM Mg²⁺,the second containing 5 mM concentrations of both Mg²⁺ and Ca²⁺. Thesequential delivery was employed here to simulate the separationpredicted by Mijangos and Diaz. The response elicited by the bimetallicsample (shown in FIG. 19) is mimicked closely by the response generatedvia the sequential delivery of two samples (FIG. 21C). It is interestinghere to note that in the instances of the monometallic samples (FIGS.21A, B) the equilibrium absorbance values of the dye core provide farmore information regarding the nature of the sample than do the temporalcomponents of the responses. In particular, the final absorbance valuesin the red channel relative to those in the green and blue channels, areuseful here for speciation. However, the utility of the ligand shell,and of the associated temporal consideration, are confirmed by thebimetallic response shown in FIG. 21C.

The moving boundary models (both mono- and bimetallic) outlined abovepredict that the progress of a metal cation through a ligand shell willbe dependent upon two factors: the diffusion coefficient of the speciesand its conditional formation constant with the immobilized ligand. Thisis confirmed by the data featured in FIG. 19 and FIGS. 21A-C, which,interestingly, present an apparent dichotomy. The plots shown in FIG.21A and FIG. 21B reveal that the EDTA shell yields almost identicalt_(L) values for Ca(NO₃)₂ and Mg(NO₃)₂. Intuitively, this suggests thatthe immobilized ligand does not appreciably discriminate between the twospecies. However, the “separation” of the bimetallic sample in FIG. 19,indicates that the EDTA shell does in fact discriminate between Ca²⁺ andMg²⁺. Given the similar diffusion coefficients of the two species,(Ca²⁺: 0.792×10⁻⁵ cm²s⁻¹; Mg²⁺: 0.706×10⁻⁵ cm²s⁻¹; measured in aqueoussolutions at 25° C.) these data suggest that when delivered individuallythe cations' progress through the matrix is governed by their diffusioncoefficients. On the other hand, the discrimination observed in thebimetallic sample may then be attributed to the ligand's preferentialbinding of Ca²⁺ over Mg²⁺. In solution, the formation constants ofEDTA-Ca²⁺ complexes are typically two orders of magnitude greater thanthose of EDTA-Mg²⁺ complexes. While the consideration of both diffusionand formation constants may greatly hamper facile rationalization ofcomplex responses, the added degree of molecular level informationcontained within the response is welcome.

The application of pattern recognition is useful for the analyses ofcomplex mixtures with cross-reactive sensor arrays. It is oftendesirable to demonstrate trends within simple multi-analyte systems.This is useful not only as proof-of-concept data, but, more importantly,it often provides insight into the workings of the array, allowing theuser to make intelligent decisions regarding the choices of patternrecognition techniques and their application to the data. To this end,an array of ligand shell particles was assembled and its responses tobinary mixtures of MgCl₂ and Ca(NO₃)₂ were examined. Interest insimultaneous analyses of Mg²⁺ and Ca²⁺ derives from a unique combinationof their biological relevance, and their inherent similarity. Indeed, asone species often interferes with detection of the other, theircoexistence within biological samples has historically challengedanalysts. The concentrations of each metal salt varied from 1 to 5 mM in1 mM increments, for a total of 25 combinations. FIG. 22 features theabsorbance vs. time responses of an EDTA-ALZC particle to a subset ofthese solutions. In each of the plots depicted in FIG. 22, the top linerepresents the green absorbance, the middle line represents the redabsorbance, and the top line represents the blue absorbance. In theresponses presented here, a number of trends are evident. At a glance,it can be seen that there is a significant delay prior to each response,and that many of the responses appear to occur in two steps. It can alsobe seen that the temporal development of these steps varies considerablywith the concentrations of the individual components. Furthermore, basedon the spectral characteristics of the individual steps, it againappears that Mg²⁺ reaches the dye core before Ca²⁺. It is alsointeresting to note that the net color changes in these responses havelittle if any variation.

For each of the 25 binary mixtures introduced to the array, two temporalcomponents of the EDTA-ALZC particle's response were quantifiedmanually: the initial delay prior to the dye's observed response (termed“primary delay”) and the duration between initial observation of thedye's response and the observation of a second step in the dye'sresponse (termed “secondary delay”). FIGS. 23A-B features plots of theparticle's primary (FIG. 23A) and secondary (FIG. 23B) delays vs. Mg²⁺and Ca²⁺ concentration. No secondary delay was recorded for solutionsthat did not elicit discernable steps. Interestingly, two differentconcentration dependent trends are evident in these plots. Increasingthe concentration of either metal decreases the primary delay, whereasthe secondary delay increases with increasing Mg²⁺ concentrations butdecreases with increasing Ca²⁺ concentrations. In this case, thesetrends are directly applicable to determining the concentrations of thetwo species, even without further data processing.

In another embodiment, particles were prepared having an indicator in aninner core of the particle, and having an amino acid, peptide, or othernitrogen containing ligands, coupled to the exterior region of theparticle. The amino acid was selected based on the ability of the aminoacid to complex with various metal cations. Each particle was exposed toa variety of metal salts to determine the amount of time it takes forthe metal cation to reach the core and induce a colormetric change inthe indicator. The time required to induce a change in the indicator isreferred herein as the “breakthrough” time. Table 1 shows thebreakthrough times for various metals with various particles. The“conjugate” column indicates the molecule bound to the exterior region.Two runs were performed for Hg, Pb, Cu, and Ni, only one runs wasperformed for Cd. TABLE 1 CONJUGATE Cd²⁺ Hg²⁺ Pb²⁺ Cu²⁺ Ni²⁺ 1-Cysteine1562 s 945 s, 952 s 799 s, 803 s n/a 1182 s, 1195 s 1-Histidine  284 s589 s, 589 s  80 s, 98 s 1173 s, 1176 s 1158 s, 1687 s EDTA  492 s 360s, 403 s 267 s, 275 s  315 s, 411 s  211 s, 438 s

Table 2 shows the breakthrough times for Hg with various particles. The“conjugate” column indicates the molecule bound to the exterior region.The times shown are an average of four runs for each conjugate. TABLE 2CONJUGATE AVERAGE BREAKTHROUGH TIME 1-Cysteine 831 ± 4 Cysteinedipeptide 989 ± 5 Cysteine tripeptide 1317 ± 6  1-Histidine 604 ± 3 EDTA577 ± 6

FIG. 24 shows a breakthrough curve characteristic of two metals passingthrough a single particle. Here we show two separate particles(histidine conjugated and cysteine conjugated) with a solution of 5 mMCd and 5 mM Hg. Utilizing HSAB theory, we expect that Cd will bind moretightly to the histidine conjugated particles than to a cysteineconjugated particle. We would expect the opposite phenomenon for Hg.This data and subsequent control studies demonstrates these basicprinciples as well as the separation of two metals on a single 200 umparticle.

The selection of the appropriate ligands for coupling to the exteriorregion of a multi-shell particle may be performed using combinatorialmethodologies. One method used to determine the presence of an analyteis a displacement assay. In one embodiment, particles that areconjugated with a receptor on the exterior region are reacted with theanalyte of interest. Those particles with an exterior region with astrongly chelating peptide will remain fluorescent since the metal willnot reach the core in a specified time period; whereas, the metal willquickly pass into the core of particles with shells that are weaklychelating and quench the fluorescence. By stopping the influx of theanalyte and then analyzing the library, the particles with a stronglychelating shell can be separated. In embodiments where the exteriorregion is coupled with peptides, the peptides may be removed from theparticle and separated using Edmond sequencing techniques.

In one embodiment, a plurality of particles having a variety of peptidescoupled to their outer shell may be produced. The inner core of all ofthe particles may have the same indicator (e.g., Fluorexon). For peptidelibraries up to 20^(n) different particles may be produced in a library,where n is the number of amino acids in the peptide chain. Because ofthe large number of different particles in these libraries, the testingof each individual particle is very difficult.

When a plurality of particles is used, the analyte will bind to theparticles at various strengths, depending on the receptor coupled to theparticle. The strength of binding is typically associated with thedegree of color or fluorescence produced by the particle. A particlethat exhibits a strong color or fluorescence in the presence of theindicator has a receptor that strongly binds with the indicator. Aparticle that exhibits a weak or no color or fluorescence has a receptorthat only weakly binds the indicator. Ideally, the particles which havethe best binding with the indicator should be selected for use overparticles that have weak or no binding with the indicator. In oneembodiment, a flow cytometer may be used to separate particles based onthe intensity of color or fluorescence of the particle. Generally, aflow cytometer allows analysis of each individual particle. Theparticles may be passed through a flow cell that allows the intensity ofcolor or fluorescence of the particle to be measured. Depending on themeasured intensity, the particle may be collected or sent to a wastecollection vessel. For the determination of an optimal particle forinteraction with an indicator, the flow cytometer may be set up toaccept only particles having an color or fluorescence above a certainthreshold. Particles that do not meet the selected threshold, (i.e.,particles that have weak or no binding with the indicator) are notcollected and removed from the screening process. Flow cytometers arecommercially available from a number of sources.

After the particle library has been optimized for the indicator, theparticles that have been collected represent a reduced population of theoriginally produced particles. If the population of particles is toolarge, additional screening may be done by raising the intensitythreshold.

The collected particles represent the optimal particles for use with theselected analyte and indicator. The identity of the receptor coupled tothe particle may be determined using known techniques. After thereceptor is identified, the particle may be reproduced and used foranalysis of samples.

EXAMPLES

Materials

Polystyrene-polyethylene glycol (PS-PEG) graft copolymer microspheres(≈130 μm in diameter when dry and 230 μm when hydrated) were purchasedfrom Novabiochem. Normal amine activation substitution levels for theseparticles were between 0.2 and 0.4 mmol/g. Commercial-grade reagentswere purchased from Aldrich and used without further purification exceptas indicated below. Fluorescein isothiocyanate was purchased fromMolecular Probes. All solvents were purchased from EM Science and thoseused for solid-phase synthesis were dried over molecular sieves.Methanol was distilled from magnesium turnings.

Immunoassays were performed using carbonyl diimidazole (CDI) activatedTrisacryl® GF-2000 available from Pierce Chemical (Rockford, Ill.). Theparticle size for this support ranged between 40 and 80 μm. The reportedCDI activation level was >50 μmoles/mL gel. Viral antigen and monoclonalantibody reagents were purchased from Biodesign International(Kennebunk, Me.). Rhodamine and Cy2-conjugated goat anti-mouse antibodywas purchased from Jackson ImmunoResearch Laboratories, Inc. (WestGrove, Pa.). Antigen and antibody reagents were aliquoted and stored at2-8° C. for short term and at −20° C. for long term. Goat anti-mouseantibody was diluted with glycerol (50%)/water (50%) and stored at −20°C.

Agarose particles (6% crosslinked) used for the enzyme-based studieswere purchased from XC Particle Corp. (Lowell, Mass.). The particleswere glyoxal activated (20 μmoles of activation sites per milliliter)and were stored in sodium azide solution. Agarose particle sizes rangedfrom 250 μm to 350 μm.

Alizarin complexone (ALZC), N,N-diisopropylethylamine (DIEA),1,3-dicyclohexylcarbodiimide (DCC, 1.0 M in dichloromethane),N,N-dimethylformamide (DMF), 9-fluorenylmethoxycarbonyl chloroformate(Fmoc), ethylenediaminetetraacetic acid dianhydride (EDTAan),diethylenetriaminepentaacetic acid dianhydride (DTPAan),nitrilotriacetic acid (NTA), acetic anhydride (AC₂O), triethylamine(TEA), and piperidine were all purchased from Aldrich and used withoutany further purification. NovaSyn TG amino resin LL (TG-NH₂) waspurchased from NovaBiochem (San Diego, Calif.). The amine concentrationwas listed by the manufacturer as 0.29 mmol/g. The average diameter waslisted as 130 μm when dry and was measured as ˜170 μm in aqueoussolutions buffered at pH 9.8 with 50 mM alanine. The following metalsalts were used in making the metal cation solutions: Ni(NO₃)₂.6H₂O,Zn(NO₃)₂.6H₂O, and Pb(NO₃)₂ Ca(NO₃)₂.4H₂O, Mg(NO₃)₂.6H₂O, andMgCl₂.6H₂O. Ca²⁺ and Mg²⁺ solutions were buffered at pH 9.8 with 50 mMalanine. Solutions of heavier metals were buffered at pH 4.8 with 50 mMacetate.

Particle Preparations

All final functionalized PS-PEG copolymer microsphere batches (resin)were dried under high vacuum for at least twelve hours. The resin waswashed thoroughly before and after each coupling reaction on the solidphase using a rotary evaporator motor to tumble the reaction vessel inan oblong fashion (shaking), for a specified period of time (i.e., the“1×1” notation refers to one wash for one minute before the solvent wasdrained).

Indicator Immobilization via Amide Linkages

Amino-terminated polystyrene-polyethylene glycol graft copolymer resin(0.20 g, 0.29 mmol/g, 0.058 mmol) was placed in a solid phase reactionvessel and washed with 1×1 minute dichloromethane, 2×5 minutesN,N-dimethyl formamide (DMF), and 2×2 minutes dichloromethane. While theresin was being washed, an oven-dried round-bottom flask was chargedwith dicyclohexylcarbodiimide (DCC) (0.059 g, 0.29 mmol, 5 eq.) andhydroxybenzotriazole (HOBt) (0.039 g, 0.29 mmol, 5 eq.) in 8 mL DMF andcooled in an ice-bath. To this mixture, alizarin complexone (0.20 g,0.29 mmol, 5 eq.) was added and the solution stirred at 0° C. for 30minutes. After completing the washes of the resin, this solution wasfiltered and added to the resin. The heterogeneous system was allowed toshake for 2-15 hours at 25° C. At the end of this time, the couplingsolution was removed and the resin was washed with 2×2 minute DMF, 1×2minute dichloromethane, 1×2 minute methanol, 1×5 minute DMF and 1×1minute dichloromethane. A small portion of this resin was then subjectedto a quantitative ninhydrin (Kaiser) test to assay for the presence ofprimary amines, using Merrifield's quantitative procedures. Variousindicator substitution levels were used as required for the desiredassays.

Other dyes such as xylenol orange (Sigma), calconcarboxylic acid(Aldrich) and thymolphthalexon (Aldrich) were conjugated to the resinparticles using similar protocols as described above.

Indicator Immobilization via Thiourea Linkage

Once the resin (0.075 g, 0.30 mmol/g, 0.0218 mmol) had been completelywashed, fluorescein isothiocyanate (0.034 g, 0.087 mmol, 4 eq.) in 5 mLdichloromethane and 5 mL DMF was added to it. Two different levels ofdye loading were created so as to service the specific needs of thecolorimetric and fluorescence-based measurements. If the resin was to beused for colorimetric studies, it was allowed to shake in an oven at 55°C. for 1-5 days. The subsequent work-up of washes was followed aspreviously mentioned. If a positive ninhydrin test was obtained, theresin was resubmitted to the reaction conditions until ninhydrin gave anegative result. Resin designated for fluorescence studies was shaken at25° C. only for 1-3 days as lower dye loading was needed. A quantitativeninhydrin test was then performed to assess the level of substitution. Alow loading volume was required to minimize fluorescence self-quenching.

Acetylated Resin

Prewashed resin (0.10 g, 0.29 mmol/g, 0.029 mmol) was treated withacetic anhydride (1.5 mL, 15.9 mmol, 548 eq.) and triethylamine (0.034g, 7.2 mmol, 248 eq.) in 5 mL dichloromethane. After 30 minutes ofshaking at 25° C., the reaction mixture was removed and the resin waswashed (as described above). A ninhydrin test produced a negativeresult.

Antigen Immobilization for Viral Immunoassays

Hepatitis B surface antigen (HbsAg) was coupled to the CDI-activatedTrisacryl support in the following manner: 20 μL of a 50% (by volume)particle slurry was pipetted into a 0.6 mL microcentrifuge tube. Thenumber of moles activated CDI sites per mL particle slurry wasdetermined and reacted with HBsAg in a 1:3000 ratio (1 mole protein:3000 moles CDI sites). To the microcentrifuge tube was added 500 μL of asolution of phosphate buffered saline at pH 8. The resulting reactionmixture was allowed to react overnight at RT with shaking. Similarprocedures were performed with HIV gp 41/120 and influenza A antigens.

Enzyme Immobilization

Diaphorase was immobilized onto porous cross-linked agarose particles(XC Particle Corp., Lowell, Mass.). The particles were purchasedpre-activated with glyoxal groups. A standard procedure for enzymeimmobilization follows. About 2 mg lyophilized diaphorase was dissolvedinto 1.00 ml solution of 200 mM phosphate buffer at pH 7.00. To 1.5 mlEppendorf tube, 100 μl of fresh particles were added and the supernatantwas removed with a pipette. To the particles was added 500 μL of 200 mMphosphate buffer (pH 7.00). A 50 μl aliquot of the diaphorase suspensionwas combined to the particle slurry and finally 20 μl of a 0.75 mMsolution of sodium cyanoborohydride was added to the mixture. Theresulting sample was then shaken at the lowest speed on a Vortex Genieovernight. The supernatant was removed the next day and the particleswere washed with 200 mM phosphate buffer (pH 7.00) twice before use.

Array Preparation

Individual microspheres were placed into chemically etched microcavitiespatterned in a square array on 4-inch single crystal (100) doublepolished silicon wafers (˜220 μm thick) using a micromanipulator on anx-y-z translator. The cavities were prepared using bulk KOH anisotropicetching of the silicon substrate. To mask the substrate during the KOHetch, a silicon nitride layer was prepared using a low pressure chemicalvapor deposition (LPCVD) technique. Removal of the mask layer from oneside of the silicon substrate was carried out by protecting the otherside with photoresist and plasma etching (CF₄ and O₂ at 100 watts) theSi₃N₄ layer. The silicon substrate was etched anisotropically using a40% KOH solution (Transene silicon etchant PSE-200) at 100° C. The etchrate of the (100) silicon was about 1 μm/min at 100° C. Successfulpatterning requires that a highly stable temperature be maintainedthroughout the etch process. After completion of the KOH etch, thenitride masking layer was completely removed from both sides of thesilicon substrate using plasma etching. To improve surface wettingcharacteristics, the completed device was soaked in 30% H₂O₂ for 15 to20 min. to form a thin SiO₂ layer on the surface of the silicon.

Flow Cell Construction

Construction of the flow cell began with the machining of two Teflonframes. Drilling a hole through the Teflon allowed for the penetrationof the interior of the frame with segments of the fluid delivery tubing.A siloxane polymer casing was then poured around each frame-tubingensemble. Two different molds were used when pouring the siloxane resin.The mold for the upper layer coated the Teflon with a thin layer ofresin and filled in the center of the frame, but left a shallowindentation in the center (at the end of the PEEK tubing) which servedas a reservoir. The lower mold yielded an almost identical piece, exceptthat it had two concentric indentations: one to hold the chip in placeand a second to serve as a reservoir below the array of particles. Thechip was then placed between the two siloxane/Teflon layers and themulti-layered structure was held together by an aluminum casing. Theresulting assembly was a cell with optical windows above and below thechip and a small exchange volume (˜50 μL) capable of handling flow ratesas high as 10 mL/min.

Fluid Delivery

Solutions were typically introduced into the flow cell using an AmershamPharmacia Biotech ÄKTA Fast Protein Liquid Chromatograph (FPLC). Thisinstrumentation was used without placement of in-line chromatographiccolumns and served as a precise, versatile and programmable pump. TheFPLC instrumentation included a number of on-board diagnostic elementsthat aided in the characterization of the system. The siloxane layersmentioned above were used to hold the chip in place and also providedfluid coupling to the delivery tubing.

Particles within the sensor array were exposed to analytes as solutionwas pumped into the upper reservoir of the cell, forced down through thewells to the lower reservoir and out through the drain. The cell wasdesigned specifically to force all introduced solution to pass throughthe wells of the array. The FPLC unit utilized here was able to drawfrom as many as 16 different solutions and was also equipped with aninjection valve and sample loop, allowing for a wide range of fluidsamples to be analyzed.

Microscope and CCD Camera

The flow cell sat on the stage of an Olympus SZX12 stereo microscope.The microscope was outfitted for both top and bottom white illumination.The scope also had a mercury lamp for fluorescence excitation. Removablefilter cubes were inserted to control the excitation and emissionwavelengths. The array was observed through the microscope optics andimages were captured using an Optronics DEI-750 3-chip charge coupleddevice (CCD) (mounted on the microscope) in conjunction with an IntegralTechnology Flashbus capture card.

Software

Image Pro Plus 4.0 software from Media Cybernetics was used on a DellPrecision 420 workstation to capture and analyze images. Solutionintroduction, image capture and data extraction were completed in anautomated fashion. The FPLC was controlled by Unicorn 3.0 software(Amersham Pharmacia Biotech).

Total Analysis System

Automated data acquisition and analysis was completed typically as amulti-step process. Initially, methods were composed within the FPLC'ssoftware. The method was laid out as a timeline and controls the fluiddelivery (i.e. flow rate, solution concentration, timing of sampleinjections, etc.). Similarly, macros within the imaging software wereused to control the timing and frequency of data capture. Typically, rawdata was in the form of a movie, or a sequence of images. After asequence had been captured, there was a pause in the automation, duringwhich time the user would define specific areas of interest to beanalyzed (i.e., the central regions of the particles) and also specifywhat information was to be extracted (i.e., average red, green, and blueintensities). A macro would then proceed through the sequence of imagesapplying the same areas of interest to each frame and exporting theappropriate information to a pre-formatted spreadsheet.

Other Instrumentation

The ¹H and ¹³C NMR spectra were obtained in CDCl₃ solvent solution thatwas used as purchased. Spectra were recorded on a Varian Unity 300 (300MHz) Instrument. Low- and high-resolution mass spectra were measuredwith Finnigan TSQ70 and VG analytical ZAB2-E mass spectrometers,respectively. Immunoassay reagent quality control tests were performedon a Molecular Devices SpectraMax Plus UV/VIS microplate reader and aMolecular Devices SpectraMax Gemini XS Spectrofluorometer microplatereader.

Coupling of Antibodies to Particles Using a Sensor Array System

In an embodiment, different particles were manufactured by coupling adifferent antibody to an agarose particle. The agarose particleparticles were obtained from XC Corporation, Lowell Mass. The particleshad an average diameter of about 280 μm The receptor ligands of theantibodies were attached to agarose particle particles using a reductiveamination process between a terminal resin bound glyoxal and an antibodyto form a reversible Schiff Base complex which can be selectivelyreduced and stabilized as covalent linkages by using a reducing agentsuch as sodium cyanoborohydride. (See Borch et al. J. Am. Chem. Soc.1971, 93, 2897-2904, which is incorporated fully herein.).

Detection Methods Using a Sensor Array System

Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex were performedcalorimetrically using a CCD device, as previously described. Foridentification and quantification of the analyte species, changes in thelight absorption and light emission properties of the immobilizedparticle-analyte-visualization reagent complex were exploited.Identification based upon absorption properties are described herein.Upon exposure to the chromogenic signal generating process, colorchanges for the particles were about 90% complete within about one hourof exposure. Data streams composed of red, green, and blue (RGB) lightintensities were acquired and processed for each of the individualparticle elements.

Detection of Hepatitis B HBsAg in the Presence of HIV gp41/120,Influenza a Using a Sensor Array System

In an embodiment, three different particles were manufactured bycoupling a HIV gp41/120, Influenza A and Hepatitis B (HBsAg) antigens toa particle (FIG. 25A). A series of HIV gp41/120 particles were placedwithin micromachined wells in a column of a sensor array. Similarly,Influenza A and Hepatitis B HBsAg particles are placed withinmicromachined wells of the sensor array. Introduction of a fluidcontaining HBsAg specific IgG was accomplished through the top of thesensor array with passage through the openings at the bottom of eachcavity. Unbound HBsAg-IgG was washed away using a pH 7.6 TRIS buffersolution. The particle-analyte complex was then exposed to a fluorophorevisualization reagent (e.g., CY2, FIG. 25B). A wash fluid was passedover the sensor array to remove the unreacted visualization agent.Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcolorimetrically using a CCD device. Particles that have form complexeswith HBsAg specific IgG exhibit a higher fluorescent value than thenoncomplexed Influenza A and HIV gp41/120 particles.

Detection of CRP Using a Sensor Array System

In an embodiment, a series of 10 particles were manufactured by couplinga CRP antibody to the particles at a high concentration (6 mg/mL). Asecond series of 10 particles were manufactured by coupling the CRPantibody to the particles at medium concentration (3 mg/mL). A thirdseries of 10 particles were manufactured by coupling the CRP antibody toparticles at a low concentration (0.5 mg/mL). A fourth series of 5particles were manufactured by coupling an immunoglobulin to theparticles. The fourth series of particles were a control for the assay.The particles were positioned in columns within micromachined wellsformed in silicon/silicon nitride wafers, thus confining the particlesto individually addressable positions on a multi-component chip.

The sensor array was blocked with 3% bovine serum albumin in phosphatebuffered solution (PBS) was passed through the sensor array systemIntroduction of the analyte fluid (1,000 ng/mL of CRP) was accomplishedthrough the top of the sensor array with passage through the openings atthe bottom of each cavity. The particle-analyte complex was then exposedto a visualization reagent (e.g., horseradish peroxidase-linkedantibodies). A dye (e.g., 3-amino-9-ethylcarbazole) was added to thesensor array. Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcolorimetrically using a CCD device. The average blue responses of theparticles to CRP are depicted in FIG. 26. The particles with the highestconcentration of CRP-specific antibody (6 mg/mL) exhibited a darker bluecolor. The control particles (0 mg/mL) exhibited little color.

Dosage Response for CRP Using a Sensor Array System

In an embodiment, a series of 10 particles were manufactured by couplinga CRP antibody to the particles at a high concentration (6 mg/mL). Asecond series of 10 particles were manufactured by coupling the CRPantibody to the particles at a medium concentration (3 mg/mL). A thirdseries of 10 particles were manufactured by coupling the CRP antibody tothe particles at a low concentration (0.5 mg/mL). A fourth series of 5particles were manufactured by coupling an immunoglobulin to theparticles. The fourth series of particles were a control for the assay.The particles were positioned in columns within micromachined wellsformed in silicon/silicon nitride wafers, thus confining the particlesto individually addressable positions on a multi-component chip.

The sensor array was blocked with 3% bovine serum albumin in phosphatebuffered solution (PBS) was passed through the sensor array system.Introduction of multiple streams of analyte fluids at varyingconcentrations (0 to 10,000 ng/mL) were accomplished through the top ofthe sensor array with passage through the openings at the bottom of eachcavity. The particle-analyte complex was then exposed to a visualizationreagent (e.g., horseradish peroxidase-linked antibodies). A dye (e.g.,3-amino-9-ethylcarbazole) was added to the sensor array.Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcalorimetrically using a CCD device. The dose dependent signals aregraphically depicted in FIG. 27.

Simultaneous Detection of CRP and IL-6 Using a Sensor Array System

In an embodiment, three different particles were manufactured bycoupling Fibrinogen. CRP and IL-6 antibodies to an agarose particle. Aseries of CRP and IL-6 antibodies receptor particles, were positionedwithin micromachined wells formed in silicon/silicon nitride wafers,thus confining the particles to individually addressable positions on amulti-component chip. A series of control particles were also placed inthe sensor array. The sensor array was blocked by passing 3% bovineserum albumin in phosphate buffered solution (PBS) through the sensorarray system. Introduction of the analyte fluids was accomplishedthrough the top of the sensor array with passage through the openings atthe bottom of each cavity. The particle-analyte complex was then exposedto a visualization reagent (e.g., horseradish peroxidase-linkedantibodies). A dye (e.g., 3-amino-9-ethylcarbazole) was added to thesensor array. Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcolorimetrically using a CCD device. The average blue responses of theparticles to a fluid that includes buffer only (FIG. 28A), CRP (FIG.28B), interluekin-6 (FIG. 28C) and a combination of CRP andinterleukin-6 (FIG. 28D) are graphically depicted in FIG. 28.

This example demonstrated a number of important factors related to thedesign, testing, and functionality of micromachined array sensors forcardiac risk factor analyses. First, derivatization of agarose particleswith both antibodies was completed. These structures were shown to beresponsive to plasma and a visualization process. Second, response timeswell under one hour was found for colorimetric analysis. Third,micromachined arrays suitable both for confinement of particles, as wellas optical characterization of the particles, have been prepared.Fourth, each particle is a fill assay, which allows for simultaneousexecution of multiple trials. More trials provide results that are moreaccurate. Finally, simultaneous detection of several analytes in amixture was made possible by analysis of the blue color patterns createdby the sensor array.

In an embodiment, 35 particles were manufactured by coupling a CRPantibody to the particles. The particles were positioned in columnswithin micromachined wells formed in silicon/silicon nitride wafers,thus confining the particles to individually addressable positions on amulti-component chip.

Regeneration of Sensor Array for Performing Multiple Tests

Particles coupled to 3 mg of antibody/ml of particles of either rabbitCRP-specific capture antibody (CRP) or an irrelevant rabbit anti-H.pylori-specific antibody (CTL) are tested for their capacity to detect1,000 ng/ml of CRP in human serum in continuous repetitive runs. FIG. 29depicts data collected using a colorimetric method. Here each cycleinvolves: i) injection of 1,000 ng/ml CRP, ii) addition ofHRP-conjugated anti-CRP detecting antibody, iii) addition of AEC, iv)elution of signal with 80% methanol, v) wash with PBS, vi) regenerationwith glycine-HCl buffer and vii) equilibration with PBS. Results shownin FIG. 29 are for the mean blue absorbance values. The results showthat regeneration of the system can be achieved over to allow multipletesting cycles to be performed with a single sensor array.

Particle Preparation—Multi-Layer Particles

Preparations were performed in a custom-made fritted solid-phasereaction vessel. The body of the reaction vessel was roughly cylindricalwith a radius of ˜12 mm, a height of ˜82 mm, and a measured volume of 24mL. The top of the body had a polytetrafluoroethylene (PTFE) lined screwcap, the removal of which permitted the addition of resin and/orsolutions. The other end of the body terminated in a porous glass frit(diameter: 20 mm; porosity: coarse). Appended to the frit end of thevessel was a double oblique bore stopcock with a PTFE plug. One of thestopcock's three stems was mated to the frit, such that either of thetwo opposing sterns could be used to drain solution from the vessel. Anexample of a commercially available vessel of similar design is LABGLASSitem# LG-5000 (www.lab-glass.com). The vessel was mounted on modifiedGlasCol® mini-rotator, allowing end-over-end tumbling of the vessel.

Provided in tabular form here is the procedure used to prepare batchesiv, v and vi (see FIG. 14 and accompanying discussion). This descriptionis applicable to numerous types of multishell particle preparations.Within a given table, each row represents a single step of that specificpreparation. Each step may be characterized as either an incubation or arinse procedure. Incubations include the removal (via aspiration) of anysolution from the reaction vessel, the addition of the indicatedsolution to the reaction vessel, and the subsequent tumbling of thevessel at ˜40 rpm for the listed time interval (hours:minutes). Rinsesinclude the removal (via aspiration) of any solution from the reactionvessel followed by the addition of the indicated solution. Multiplerinses of a single solvent are condensed into a single step in thetable, with the number of rinses indicated. Additionally, entries in thethird column in each table comment on the purpose of the key syntheticsteps. The total solution volume was held consistently at 18 mL, unlessotherwise noted. It should be mentioned that incubations in excess of 3hrs represent the resin being left overnight, and that their times werebased on convenience rather than necessity. Initially, 200 mg of TG-NH₂was modified as shown below in Table 3. TABLE 3 Preparation ofMultishell Particle Batch iv Incubation Time Number of (hrs:min) RinsesSolution Composition Purpose 1× DMF  0:10 DMF  1:04 DMF  2:10 100 uLDIEA in 18 mL DMF  0:18 8 mM Fmoc, 50 uL protect exterior region DIEA in15 mL DMF  0:20 3 mM ALZC, 3 mM dye core DCC in 18 mL DMF 2× DMF 2× HCl(10 mM)  0:03 HCl (10 mM)  0:09 HCl (10 mM)  0:03 NaOH (10 mM) 1× HCl(10 mM)  0:30 NaOH (10 mM) 1× HCl (10 mM)  2:30 NaOH (10 mM) 1× HCl (10mM) 1× NaOH (10 mM) 2× H2O 3× DMF  1:12 DMF  0:15 25% piperidine in DMFcleave Fmoc  0:35 25% piperidine in DMF cleave Fmoc 1× DMF 13:42 DMF 1:53 25% piperidine in DMF cleave Fmoc 1× DMF 30:00 DMF

TABLE 4 Preparation of Multishell Particle Batch v Incubation TimeNumber of (hrs:min) Rinses Solution Composition Purpose  0:25 DMF  0:351:1:3 Ac2O:TEA:DMF acetylate exterior 1× DMF  0:05 DMF  0:12 DMF 15:15DMF  0:09 DMF 2× H2O  0:15 H2O  1:15 H2O  1:12 H2O

The resulting resin, with acetylated exterior amines and ALZC cores, wascollected and labeled as Batch v. A second aliquot of Batch iv wastreated with EDTA anhydride and then washed, as shown below in Table 5.TABLE 5 Preparation of Multishell Particle Batch vi Incubation TimeNumber of (hrs:min) Rinses Solution Composition Purpose  0:25 DMF  0:4010 mM EDTAan in 20% EDTA in exterior TEA/DMF 1× DMF  0:05 DMF  0:12 DMF15:15 DMF  0:09 DMF 2× H2O  0:15 H2O  1:15 H2O  1:12 H2O

The resulting resin, with immobilized EDTA in the exterior regions andALZC in the cores, was collected and labeled as Batch vi. Samples fromBatches v and vi were subjected to a further attempteddye-immobilization reaction in order to reveal any free amines in theexterior regions. Visual inspection indicated that no dye wassuccessfully immobilized in the outer shells of either batch.

Data Acquisition and Analysis

Arrays of multishell particles are arranged on silicon chips andsubsequently sealed in custom-built flow cells. The flow cell is readilyinterfaced with a variety of fluidic devices (i.e., pumps, valves), theprecise configuration of which is dictated by individual experiments. Inthe flow cell, the array is illuminated from below while being viewedwith a DVC 1312C CCD camera (DVC Co., Austin, Tex.) through the opticsof an Olympus SZX12 stereo microscope. For this work, image acquisitionwas controlled via LabVIEW software (National Instruments, Austin,Tex.), ensuring high temporal fidelity. Macros written and executedwithin Image Pro Plus 4.0 (Mediacybernetics) were used to generate RGBabsorbance vs. time plots for individual microspheres. The RGB effectiveabsorbance values were calculated as described earlier.

Further Improvements

In some embodiments, an optical analysis instrument for both membraneand/or sensor array particle-based measurements may be used to determinethe presence of analytes. A schematic diagram of an embodiment of aninstrument is depicted in FIG. 30. In one embodiment, an instrument mayinclude a sample collection device 700, an off-line sample processingunit 710, a fluid delivery system 720, a disposable cartridge 730, acartridge self-positioning system 740, an optical platform 750,electronics 760, power supplies 770, one or more computer processors780, and/or software 790 and/or firmware.

In some embodiments, the instrument may include one or more disposablecartridges. A disposable sample cartridge may be the chemical andbiochemical-sensing component of the analysis instrument. A cartridgemay include index-matching, molded or machined plastics, metals, glassor a combination thereof. A cartridge may also include one or morereservoirs for holding reagents, samples, and/or waste. Reservoirs maybe coupled to a cartridge via one or more microfluidic channels.

A cartridge may include one or more detection systems. As used hereinthe term “detection system” refers to a system having an analytedetection platform. Detection systems include both particle-basedanalyte detection platforms and membrane-based analyte detectionplatforms. A particle-based analyte detection platform may include aparticle-based platform includes particles configured to produce asignal in the presence of one or more analytes. The analysis and/orseparation surfaces (e.g., membrane or the like) and/or sensingparticles housed on a support member, may be used to determine thepresence of analytes. The membrane surface traps and/or separatesparticulate matter of interest (e.g., cells, microbes, small pieces oftissue, polymer, glass or metal particles, or conjugates thereof). Thesupport member includes sensing particles functionalized to react withanalytes of interest (e.g., proteins, DNA and RNA oligonucleotides,metals or other solution-phase analytes). As such, the cartridge mayhave the capability to detect both particulate matter and/orsolution-phase analytes concurrently.

In certain embodiments, the particle-based analyte detection platformmay include a supporting member that supports one or more particles.Particles may be optically encoded with one or more fluorophores,chromophores, etc. and used to identify the particle, regardless of thelocation of the particle and/or analyte. Such an encoding scheme may beused in a combination membrane/particle-based cartridge and may makemanufacture of the cartridge easier.

In an embodiment, (micron-sized) encoded particles may be placed in thefluid sample for the purpose of sample and/or reagent identification(e.g., a sample identification bar code). In operation, the membrane maybe used to trap the particles and identify the patient (perhaps inaddition to membrane-based analysis), followed by sensor-array analysis.Such particles may also be used to calibrate the instrument and/ormonitor the flow rate.

In some embodiments, a cartridge may be designed such that the cartridgeis removably positionable in an instrument. Cartridge alignment may beperformed manually or automatically using the cartridge positioningsystem. A cartridge positioning system may automatically or manuallyposition the disposable cartridge in the instrument. In certainembodiments, the disposable cartridge may be placed in the cartridgeself-positioning system prior to sample introduction. In one embodiment,a fluid delivery system may deliver reagents to a disposable cartridge.Once the disposable cartridge is placed inside the instrument, thecartridge positioning system may be used to align the one or more areasof the cartridge containing the sample to be analyzed with theinstrument's optical platform. The optical platform may acquire images(e.g., visual or fluorescent) of the sample, and/or of sample-modulatedparticle-based platforms. The images may be processed and analyzed usingsoftware, algorithms, and/or neural networks.

An instrument may be used to analyze one or more samples. A sample mayinclude one or more analytes, cells, and/or bacteria. A sample may becollected for analysis with a sample collection device. The samplecollection device may be external or internal to the instrument and maybe interfaced with the analysis instrument. In some embodiments, asample collection device obtains and delivers one or more samplesdirectly to an instrument. Depending on the type of measurement to beperformed, a sample may be transported through one of two pathways bythe sample collection device. In one application, a sample may betransported to an off-line sample-processing unit where the sample maybe manipulated. The sample may then be transported to a disposablecartridge via a fluid delivery system. In another embodiment, a samplemay be transported directly to a disposable cartridge by a samplecollection device. The disposable cartridge, including the sample, maythen be inserted into the instrument.

FIG. 31A depicts an embodiment of an optical analysis instrument. Asample collection device may be used to obtain a sample 800. A sample800 may be mixed with reagents 810 in an analysis instrument's off-linesample processing unit. The modified sample 820 may be coupled to theinstrument via a fluid delivery system 830. The instrument may includean actuator 840 that may force fluid, such as samples, reagents, and/orwaste, through the instrument. The fluid delivery system 830 may allow amodified sample 820 to pass over a reagent pad 850 positioned on acartridge 860. A buffer 860 may also flow over the reagent pad 850.Passing the modified sample 820 and/or buffer 860 may reconstitute oneor more reagents on the reagent pad 850. The modified sample may thenpass through a trap configured to remove air from the fluid. Themodified sample may then flow to a particle-based platform and/or amembrane-based platform for analysis. The cartridge 860 may beautomatically or manually aligned with the optical platform 880 foranalysis. Residual reagents, buffer, and/or sample may flow to a wastereservoir 890 for storage. A waste reservoir may be positioned in theinstrument or external to the instrument. A waste reservoir may reducehazards to operators by reducing an operator's contact with samplesand/or reagents.

The use of a sample collection device may help to limit the operator'sexposure to pathogens that may be present in the sample. Ideally, thesample collection device will have the ability to consume the portion ofthe device (e.g., a needle) that has contacted the sample. Oneembodiment of a sample collection device is a pressurized unit thatoperates analogous to a vacutainer used to collect blood samples, asdepicted in FIG. 31B. Using such a device, samples may be directlytransported from the source to the instrument without further handlingby the operator.

In some embodiments, a sample may be obtained intravenously using samplecollection device 890 including a needle and vacutainer. In operation, afilled vacutainer may be coupled or secured to the portable readerinstrument. A sharp sample collection needle, that is part of theportable reader instrument, may be actuated to pierce the vacutainer'srubber septum. The sample may then flow through the instrument foranalysis via a fluid delivery system 830 driven by an actuator 840. Asample may flow from a sample collection device to a sample reservoir900. Reagents 910 and/or buffer 870 may mix with the sample in thesample reservoir 900. The modified sample may then flow from the samplereservoir 900 to the cartridge 860 for analysis. Samples, reagents,buffers, and/or other fluids may flow from the cartridge 860 to a wastereservoir 890 after analysis.

In another embodiment, the sample may be obtained from a fingerstick orsmall incision and may be collected using a disposable pipette, as shownin FIG. 31C. A portion of a body may be brought proximate the instrumentwhere a sample collection device is positioned. A sample collectionneedle may be part of the portable reader instrument. A samplecollection device may include a disposable tip 930 and/or a filter 940.Using a disposable tip on a sample collection needle may inhibitsample-to sample cross-contamination. In some embodiments, a disposabletip may be at least partially coated with appropriate reagents. A samplemay be incubated in a disposable tip before being drawn into aninstrument. In an embodiment, a sample collection needle may include afilter and/or screen on a distal end. A filter and/or screen may inhibitthe entry of debris into an instrument, inhibit clogging or obstructionof an instrument, and/or inhibit clogging or obstruction of samplecartridge microfluidic channels.

Sample may flow from the sample collection device to the cartridge 860via a fluid delivery system 830. A sample may pass over a reagent pad850 positioned on the cartridge 860. Sample and/or buffer 870 mayreconstitute reagents on the reagent pad 850. After reacting with one ormore reagents, a sample may flow to a particle-based platform or amembrane-based platform for analysis. A cartridge 860 and/or opticalplatform 880 may be adjusted such that the optical platform is inalignment with the particle or membrane platform being analyzed. Afteranalysis, the sample may flow to a waste reservoir 890. A cartridge 860may be washed prior to analysis of the next sample. A fluid and/orbuffer 870 may flow through the cartridge 860 and into the wastereservoir 890.

In an embodiment, a sample collection device may include a disposablepipette or capillary tube. A disposable pipette may contain, or may becoated with, one or more appropriate reagents to aid in visualization.For example, a stain may aid in visualization of particles and/or cellsin a sample. A disposable pipette may also collect a precise samplevolume. It may be desirable to incubate a sample prior to analysis. Asample may be incubated in a disposable tip before being drawn into aninstrument. In one embodiment, after incubation, the sample may bedelivered to the cartridge manually using the disposable pipette. Inanother embodiment, a sample cartridge may include one or moreappropriate reagents for incubation in the sample or reagent reservoir.In some embodiments, incubation may be performed within the samplecartridge using reagents from a sample or reagent reservoir. After thesample is incubated with one or more reagents, the fluid delivery systemmay deliver a buffer solution to the sample/reagent reservoir.Delivering a buffer solution to the sample/reagent reservoir may pushthe labeled sample to a membrane in the cartridge for subsequent rinsingand sample analysis. After analysis of the sample is completed, thesample may be delivered to a waste reservoir. A waste reservoir may bepositioned in the sample cartridge, internal or external to theinstrument.

In some embodiments, a sample may be obtained from a fingerstick orsmall incision in a portion of a human body 941, as depicted in FIG.31D. In an embodiment, sample collection device 942 may include a samplereservoir. A sample collection device 942 and/or sample reservoir may beconfigured to collect a predetermined volume of a sample. A samplecollector device 942 may include a pipette. A sample collection device942 may be coupled to a cartridge 860 to deliver a sample to thecartridge. In one embodiment, a bulb 943 on a pipette may force a samplefrom a sample collection device 942 into the cartridge 860. A fluiddelivery system 830 coupled to the cartridge 860 may deliver buffer 870to a cartridge 860 and/or reagents. In an embodiment, sample, buffer,and/or reagents mix in a mixing chamber 944 of a sample reservoir in thecartridge 860. After the sample has been reacted with one or morereagents, the sample may flow into a membrane 945 and/or particle-basedplatform 946 of the cartridge 860 for analysis. Waste reservoirs 890,895 positioned in the cartridge or externally, respectively, may collectwaste from cartridge 860.

In an embodiment, a portion of a human body, such as a finger, may bepositioned proximate a sample reservoir of a cartridge. A portion of ahuman body may contact a portion of the sample reservoir. A samplereservoir may have a size that allows a predetermined volume of sampleto be collected. A cartridge sample reservoir may include a samplepick-up pad. A sample pick-up pad may be a pad that absorbs and/orcollects samples deposited on a surface of the sample pick-up pad. Asample pick-up pad may be made of an absorbent material. A samplepick-up pad may draw a sample from a portion of a human body in contactwith the sample pick-up pad to a sample reservoir. For example, a samplecollection device may make a small incision in a portion of a humanbody. The portion of the human body may be brought proximate a samplepick-up pad. Blood from the small incision may flow onto the samplepick-up pad. Blood from the sample pick-up pad may then be delivered tothe cartridge via a fluid delivery system. In an embodiment, a samplepick-up pad may include one or more anti-coagulants and/or reagents forsample labeling. A sample reservoir may include one or moreanti-coagulants and/or reagents for sample labeling.

In some embodiments, the instrument may include an off-linesample-processing unit. An off-line sample-processing unit may processsamples prior to delivery to a cartridge. An off-line processing unitmay allow sample processing including, but not limited to, incubationwith reagents, cell lyses and/or sample amplification techniques such asPolymerase Chain Reaction (PCR). Depending on the type of diagnosticassay or measurement being performed, an off-line sample-processing unitmay be bypassed and a sample may be directly delivered to a disposablecartridge.

In certain embodiments, a fluid delivery system may include meteredpumps (e.g., syringe, rotary, and/or peristaltic), valves, connectors,and/or pressure-driven actuation (e.g., roller with motorizedtranslation). A fluid delivery system may be vacuum-driven (e.g., acartridge may be under vacuum). A fluid delivery system may draw one ormore samples into an instrument, deliver one or more samples to a samplecartridge, and/or move fluids such as sample, reagents and/or buffersthrough the cartridge and other channels or fluid lines. A fluiddelivery system may deliver samples and/or other fluids to a wastereservoir after analysis. In one embodiment, a fluid delivery system maybe used to wash a cartridge after sample analysis. Fluid may be driventhrough a cartridge after a sample is analyzed by the fluid deliverysystem. The fluid may then flow from the cartridge to a waste reservoir.

FIG. 32A depicts one embodiment of a sample cartridge and its interfacewith an actuated fluid delivery system. In this example, the buffer 870,reagents 850, and/or sample 940 are contained in reservoirs. Reservoirsmay be substantially sealed reservoirs positioned in a cartridge. In anembodiment, applying pressure to a reservoir may release the contents ofthe reservoir into channels 950. Actuators 840 may press down on thefluid containing reservoirs, delivering the contents to the membrane 960and/or particle-based platform 970. FIG. 32B depicts an embodiment of anactuator 840. Actuator 840 may include a mechanism for applying pressureto one or more reagent packs 850, either individually or simultaneously.In one embodiment, actuator 840 includes an elongated member 980 that ismoved by the actuator 840 to apply pressure on one or more reagent packs850, causing the reagent packs to release one or more reagents to acartridge 860. During use, an actuator 840 may apply pressure to areagent pack 850, forcing one or more reagents in the reagent packthrough a channel 950, as depicted in FIG. 32A. Channels 950 may couplea reagent pack 850 to a membrane 960 and/or a particle-based platform970 in a sample cartridge 860. As pressure on a reagent pack 850increases, more reagent may be released from the reagent pack and into achannel 950. As depicted in FIG. 32C, reagents may flow through achannel 950 and into a sample cartridge 860. Sample and reagent may flowout the sample cartridge 860 via a channel due to actuation. Increasedpressure from actuators on buffer 870, sample 940, and/or reagent packs850 may drive fluid from the membrane 960 and/or particle-basedplatforms 970 and into a waste reservoir 890, see FIG. 32A. A wastereservoir 890 may be positioned in the cartridge 860.

FIG. 33A depicts an embodiment of a disposable cartridge includingreagent packs. During use, a sample (e.g., blood obtained from afingerstick) may be delivered to a sample reservoir 990. A reagent pack850 may deliver one or more reagents to a sample reservoir 990 byactuation. In an embodiment, an actuator may apply pressure on a reagentpack 850 and force reagent from a reagent pack through channels 950 andinto a sample reservoir 990. Reagents and a sample may react in thesample reservoir 990. In certain embodiments, further actuation maycause the modified sample, or sample reacted with reagents, into a trap1000. Trap 1000 may be a bubble trap. Trap 1000 may be designed torelease air from a fluid passing through it. Trap 1000 may substantiallyremove air from a sample flowing through a trap. Further actuation maythen push a substantially air free sample from a trap 1000 into amembrane and/or particle-based platform 1010. In a membrane and/orparticle-based platform 1010, a sample may be washed with a solutionand/or analyzed. Residual reagents and/or discarded samples may becollected and/or contained in a waste reservoir 890 positioned in thecartridge 860. Collecting reagents and/or samples in a waste reservoirmay facilitate hazard-free disposal of the cartridge.

FIG. 33B depicts an embodiment of a cartridge including reagent packs. Areagent pack may be a pad 855 including one or more reagents that havebeen dried on a surface of the reagent pad. A reagent pack may include apad with one or more reagents within the pad. In certain embodiments,reagents and/or a reagent pad may include one or more stabilizers.Stabilizers may increase reagent stability. During use, a sample may bedeposited in a sample reservoir 990. Buffer may be delivered throughfluid inlets and flow over reagent pads 855. When a buffer passes overreagent pads 855, one or more reagents may be reconstituted anddelivered to a sample reservoir 990. In one embodiment, a buffer mayreconstitute a desired reagent on a reagent pad 855. A buffer solutioncontaining the reconstituted reagents may pass into a sample reservoir990 and react with a sample. A fluid delivery system may then push thechemically modified sample (e.g., the sample reacted with one or morereagents) into a trap 1000. In the trap 1000, air may be released fromthe chemically modified sample. Further pressure or actuation may pushthe air free sample into a membrane and/or particle-based platform 1010of a cartridge 860. In a membrane and/or particle-based platform 1010, achemically modified sample may be washed and/or analyzed. Residualreagents and/or discarded samples may flow to a waste reservoir 890 toreduce hazards during disposal.

In some embodiments, a combination of reagent reservoirs, reagent packs,and/or reagent pads may be positioned in a cartridge, as depicted inFIG. 33C. Reagent packs and/or reservoirs 850 may be coupled to reagentpads 855 such that pressure on the reagent packs 850 may deliver one ormore reagents to one or more reagent pads 855. Reagents from the reagentpacks 850 may reconstitute one or more reagents on the reagent pads 855.Further actuation may force the reagents from the reagent pad to thesample reservoir 990. For example, an actuated lever may apply pressureto reagent packs and force reagent through one or more channelsconnecting one or more reagent packs and a sample reservoir. A channelmay direct reagent from a reagent pack to flow over a reagent pad. Insome embodiments, a cartridge 860 may include passive valves 1015, asdepicted in FIG. 33C. Passive valves provide a path of least resistanceto flow. Passive valves 1015 may be used to facilitate fluid flowtowards a sample reservoir 990 and/or other areas of the cartridge 860.A fluid delivery system may then push the chemically modified sample(e.g., the sample reacted with one or more reagents) into a trap 1000.In the trap 1000, air may be released from the chemically modifiedsample. Further pressure or actuation may push the air free sample intoa membrane and/or particle-based platform 1010 of a cartridge 860. In amembrane and/or particle-based platform 1010, a chemically modifiedsample may be washed and/or analyzed. Residual reagents and/or discardedsamples may flow to a waste reservoir 890 to reduce hazards duringdisposal.

In some embodiments, disposable cartridges may include reagent pads.Reagent pads may store reagents in a self-contained manner that mayprovide increased stability, reduce and/or eliminate reagent aggregationand/or precipitation (e.g., clumping) and increase effective reagentconcentrations. Increasing effective reagent concentrations may reduceresponse times for sample analysis. Disposable, self-containedcartridges may have important implications for point-of-carediagnostics, such as, not requiring refrigerated storage nor reagentpreparation and/or not requiring handling of waste material. Cartridgesmay allow fast and inexpensive diagnostics to be transported to andperformed in situations where time is critical.

In some embodiments, a reagent capsule including one or more reagentsmay be coupled to a cartridge. Reagent capsule may include liquid and/ordried (e.g., reagents in solid or powder form) reagents. In oneembodiment, a reagent pad with dried reagent on the pad may bepositioned in the reagent capsule. FIG. 33D depicts an exploded view ofan embodiment of a reagent capsule 1018 coupled to a cartridge 860including membrane and particle-based platform analysis regions. Acartridge 860 may include a top portion 861 and a bottom portion 862. Areagent capsule 1018 may be coupled to the cartridge 860 such thatchannels 950 coupled the reagent capsule to a trap, particle-basedplatform portion 860, and/or membrane portion 870 of the cartridge. FIG.33E depicts an embodiment of a reagent capsule 1018 coupled to acartridge 860 including membrane 870 and particle 860 basedparticle-based platform analysis regions. A sample may enter a reagentcapsule 1018 via a fluid connection line and flow via channels 950 inthe cartridge 860 to the particle-based platform 860 and/or membrane 870analysis regions.

In some embodiments, a cartridge may include reagent delivery systems,such as a reagent pack, a reservoir containing reagent, and/or a regentpad. In some embodiments, a cartridge includes a reagent delivery systemthat includes a reagent pack and reagent pad. During use, a sample maybe deposited in a sample reservoir and reagents may be delivered to thesample reservoir by actuation. In one embodiment, an actuator may applypressure to a reagent pack and force reagent through a channel, over areagent pad and into the sample reservoir where the reconstitutedreagents react with the sample. Further actuation may cause thechemically modified sample into a trap where substantially all of theair in a sample may be released. The chemically modified, air freesample may be forced by actuation onto a membrane and/or aparticle-based platform of a cartridge. In a membrane and/or aparticle-based platform of a cartridge, a sample may be washed and/oranalyzed. Residual reagents and/or sample may flow into a wastereservoir after analysis to reduce the risk of hazard during disposal.

FIG. 34 depicts another embodiment of a fluid delivery system. In thisexample, the system may be primed and filled with buffer 870. Pump 1020may draw sample (which may or may not contain reagents) into the sampleneedle 1030, through the three-way valve 1040 and into the sample loop1050. The valve 1040 is then switched and the pump 1020 pushes thesample through the valve and into the sample cartridge 1060, followed bya buffer wash. After sample analysis, the sample is pushed to a wastereservoir 890 and the system is washed with buffer 870.

FIG. 35 depicts another embodiment of a fluid delivery system. In thisexample, a metered (e.g., controlled volume) syringe pump 1070 may pushand pull fluids through the system. In operation, a capillary 1080filled with sample may be inserted into the sample cartridge 1060. Thecartridge 1060 may be “quick” connected to a fluidics bus 1090, whichmay at least partially seal the system. Quick connecting the cartridge1060 to the fluidics bus 1090 may seal the system. The system may beprimed and filled with buffer 870 through lines 2000 and 2010. Usingline 2020, the sample may be pushed into a trap 2030. In the trap 2030,the sample may be diluted with buffer 870 and air bubbles may bereleased. Line 2010 may be used to draw a known volume of the dilutedsample into the detection region 2040. Alternatively, Line 2000 may drawsample into the sample loop 1050 and the sample is pushed into thedetection region 2040 via line 2010. A four-way valve may couple fluidlines and the sample loop. After sample analysis, the system may bewashed with buffer 870 and waste may be delivered to a waste reservoir890 using line 2050.

FIGS. 36A-B depicts another embodiment of a fluid delivery system. FIG.36A depicts a schematic drawing of the fluid delivery system. In thisembodiment, a metered (e.g., controlled volume) syringe pump 1070 maypush and pull fluids through the system. In operation, a capillary 1080filled with sample may be inserted into the sample cartridge 1060. Thecartridge 1060 is “quick” connected to a fluidics bus 1090, which sealsthe system. The system may be primed and filled with buffer 870 throughlines 2060 and 2070. Using line 2060, the entire sample may be drawninto the sample loop 1050 (see magnified view of sample loop, FIG. 36B).The first one-third of the sample (a) may then be pushed back into line2060 as waste to remove air bubbles from the sample. The secondone-third of the sample (b) may be pushed into the detection region 2040of the cartridge 1060 using line 2070. The last one-third of the samplemay be pushed into line 2060 as waste to remove air bubbles from thesample. Since the pump 1070 may be metered, this method provides volumecontrol for sample delivery, without the need for a trap. After sampleanalysis, the system is washed with buffer 870 and waste is discardedthrough line 2080 into a waste reservoir 890.

In some embodiments, a cartridge self-positioning system may perform twofunctions. First, the system may be used to align (manually orautomatically) the area(s) of the cartridge containing the sample to beanalyzed with the instrument's optical platform Second, theself-positioning system may reposition the cartridge such that multipleareas of the sample may be analyzed in sequence.

A cartridge self-positioning system may include at least two components,as shown in the embodiment of a cartridge self position system depictedin FIG. 37. One component is an apparatus 2090 that may hold or securethe cartridge 1060 in place. An example of such is an apparatus thatfunctions analogous to a computer disk mount. In operation, such adevice would accept and/or eject a disposable cartridge into/out of theanalysis instrument.

A second component of the cartridge self-positioning system may behardware, software, and/or firmware capable of registering and verifyingthe position of the disposable cartridge in relation to the opticalcomponents of the analysis instrument. For example, positionregistration hardware may be comprised of an x- and/or y-motor-driventranslation stage in which position is tracked by counting the motor'ssteps to or from a home position. Alternative embodiments of positionregistration hardware include, but are not limited to: a motorizedmicrometer or actuator, a piezo-electric actuator coupled to an opticalpositioning device, an encoder wheel gear monitored by a sensor, and/ora manual translation stage or micrometer.

An instrument may include one or more optical platforms. An instrument'soptical platform may acquire images of a sample, and/or ofsample-modulated detection regions. An optical platform may translatethe acquired images into meaningful values. Images, in some embodiments,may include captured spectroscopic changes within the optical platform.In one embodiment, components of an optical platform may include one ormore light sources, one or more lenses, one or more dichroic mirrors,one or more photodetectors, one or more emission filters, and/or one ormore excitation filters.

The one or more light sources may include: a collimated, monochromaticlight source, such as a diode laser; a white light source, such as atungsten-halogen lamp; and/or light emitting diodes (LEDs). Optionally,one or more light sources may be modulated using a transistor-transistorlogic (TTL) pulse, an electronic shutter and/or an on/off switch. Theone or more light sources may emit light suitable for the excitation ofone or more reporter or encoding labels present in the sample and/or onparticles contained within the device (e.g., fluorophores; chromophores;luminophores such as single dyes, tandem or conjugate dyes; particles;and/or a combination or multiplex thereof). The excitation of eachspecies may cause one or more spectroscopic changes, such as intensity,lifetime, spectral characteristics, and/or polarization. An opticaldetector may include one or more detectors. Detectors (e.g., an arraydetector such as a charge-couple device camera) may measure theresulting properties of the excitation of each species. One or moreprocessors equipped with software may translate each measured propertyto a meaningful value.

In one embodiment, shown in FIG. 38A, an optical platform may include alight source 3000, focusing lenses 3010, at least one excitation filter3020, an electronic shutter 3030, a dichroic mirror 3040, at least oneemission filter 3050, and/or an array detector 3060. In one operation,the sample cartridge 1060 containing sample reacted with one or morefluorescent reporter labels, may be placed in a cartridge positioningsystem 3070. The positioning system 3070 aligns the sample area with theoptical path. Light from the excitation source 3000 may be collimatedwith a lens 3010, filtered to the appropriate wavelength, passed throughan open shutter 3030, reflected 90° by a (long pass or multi-bandpass)dichroic mirror 3040 and focused onto the sample using a lens 3010. Theexcitation light 3000 may excite one or more fluorophores present in thesample. The fluorescence emission from excited fluorophores may becollected by a 3010 lens and transmitted through the dichroic mirror3040, filtered 3050 to the appropriate wavelength(s) and imaged with adetector 3060, such as a CCD camera. Fluorescence images may beprocessed and a meaningful value may be reported to an operator. Whilethe above description is specific for fluorescent changes, it should beunderstood that the system may be modified to capture any kind ofspectroscopic change.

In some embodiments, a light emitting diode (LED) assembly may be usedin place of a light source in an optical system. An embodiment of an LEDassembly is depicted in FIG. 38B. An exploded view of the LED assemblydepicted in FIG. 38B is depicted in FIG. 38C. The LED assembly 3000 mayinclude a heat sink 3080, a LED 3090, a mount 4000, a filter 4010, alens tube 4020, and a focusing lens 4030.

In a second embodiment, depicted in FIG. 39, the optical platformincludes three LED light sources 4040, 4050, 4060 (e.g., blue, green andred); focusing lenses 3010 for each of the LED lights; three excitationfilters 3020; three light source modulation units (e.g., electronicshutters) 3030; three dichroic mirrors 4070, 4080, 4090; at least oneemission filter 3050; and an array detector 3060. In one embodiment, asample cartridge 1060 containing sample reacted with one or morefluorescent reporter labels may be placed in a cartridge positioningsystem 3070. The cartridge positioning system 3070 aligns the samplearea with the optical path. Blue light from excitation source 4040 maybe collimated with a lens 3010, filtered to the appropriate wavelength,passed through an open shutter 3030, reflected 90° by a (long pass)dichroic mirror 4070 and focused onto the sample using a lens 3010. Theblue excitation light may excite blue-excited fluorophores present inthe sample. The fluorescence emission from the blue-excited fluorophoresmay be collected by a lens 3010, transmitted through dichroic mirrors4070 and 4080 (multi-bandpass dichroic), filtered 3050 to theappropriate wavelength(s), and imaged with a detector 3060, such as aCCD camera. Next, green light from excitation source 4050 may becollimated with a lens 3010, filtered 3020 to the appropriatewavelength, passed through an open shutter 3030, reflected 90° bydichroic mirror 4090 (long pass), reflected 90° by dichroic mirror 4080(multi-bandpass dichroic), transmitted through dichroic mirror 4070(long pass) and focused onto the sample using a lens 3010. The greenexcitation light may excite green-excited fluorophores present in thesample. The fluorescence emission from the green-excited fluorophoresmay be collected by a lens 3010, transmitted through dichroic mirrors4070 and 4080 (multi-bandpass dichroic), filtered to the appropriatewavelength(s), and imaged with detector 3060. Next, red light fromexcitation source 4060 may be collimated with a lens 3010, filtered tothe appropriate wavelength, passed through an open shutter 3030,transmitted through dichroic mirror 4090 (long pass), reflected 90° bydichroic mirror 4080 (multi-bandpass dichroic), transmitted throughdichroic mirror 4070 (long pass), and focused onto the sample using alens 3010. The red excitation light may excite red-excited fluorophorespresent in the sample. The fluorescence emission from the red excitedfluorophore may be collected by a lens 3010; transmitted throughdichroic mirrors 4070 and 4080 (multi-bandpass dichroic); filtered 3050to the appropriate wavelength(s); and imaged with a detector 3060. Thethree-color fluorescence images may then be processed and a meaningfulvalue may be reported to the operator. While the above description isspecific for fluorescent changes, it should be understood that thesystem may be modified to capture any kind of spectroscopic change.

In an embodiment, shown in FIG. 40, images of multiple colors may beacquired simultaneously. In this embodiment, the optical platformincludes two diode laser light sources (e.g., green and red) 5000, 5010;focusing lenses 3010; two light source modulation units (e.g.,electronic shutters) 5020, 5030; three dichroic mirrors 5040, 5050,5060; two emission filters 5070, 5080 and two array detectors 5090,6000. A sample cartridge 1060 containing sample reacted with one or morefluorescent reporter labels may be placed into the cartridge positioningsystem 3070. The cartridge positioning system 3070 may align the samplearea with the optical path. Green light from excitation source 5000 maybe focused with a lens 3010, passed through an open shutter 3030,reflected 90° by (long pass) dichroic mirror 5040, and focused onto thesample using a lens 3010. The green excitation light may excitegreen-excited fluorophores present in the sample. The fluorescenceemission from the green-excited fluorophores may be collected by a lens3010, transmitted through dichroic mirrors 5040 (long pass), 5050(dual-bandpass dichroic), reflected 90° by dichroic mirror 5060 (longpass), filtered 3050 to the appropriate wavelength and imaged withdetector 5090. Simultaneously, red light from excitation source 5010 maybe focused with a lens 3010, passed through an open shutter 3030,reflected 90° by dichroic mirror 5050 (dual-bandpass dichroic),transmitted through dichroic mirror 5040 (long pass), and focused ontothe sample using a lens 3010. The red excitation light may excitered-excited fluorophores present in the sample. The fluorescenceemission from the red-excited fluorophores may be collected by a lens3010; transmitted through dichroic mirrors 5040 (long pass); 5050(dual-bandpass dichroic) and 5060 (long pass); filtered 3060 to theappropriate wavelength; and imaged with detector 6000. The two-colorfluorescence images may be processed simultaneously and a meaningfulvalue may be reported to the operator. While the above description isspecific for fluorescent changes, it should be understood that thesystem may be modified to capture any kind of spectroscopic change.

FIG. 41 is a schematic drawing of an embodiment of an optical system inwhich the light sources are laser diodes. A sample may be delivered tothe sample cartridge 1060 using a syringe pump-based fluid deliverysystem 6010. Light from laser diode 6020 may be transmitted throughdichroic mirror 6030, optionally filtered, reflected off dichroic mirror6040, and focused onto the sample. Fluorescence from the sample iscollected by the lens; reflected off dichroic mirror 6040; filtered tothe appropriate wavelength; and imaged onto a detector 6050.Simultaneously, or in sequence, light from laser diode 6060 may bereflected off dichroic mirror 6030, optionally filtered, reflected offdichroic mirror 6040, and focused onto the sample. Fluorescence from thesample may be collected by a lens, reflected off dichroic 6040, filteredto the appropriate wavelength, and imaged onto a detector 6050. Whilethe above description is specific for fluorescent changes, it should beunderstood that the system may be modified to capture any kind ofspectroscopic change.

Optionally, an optical platform may include one or more optical fibers(e.g., single-core optical fibers, imaging fibers, bifurcated fibers, ora group thereof). Optical fibers may carry excitation light to the oneor more labels present in the sample and may carry the emittedfluorescence properties to one or more detectors. Additionally, multiplefibers may be employed to image multiple regions of the sample areasimultaneously, thus eliminating the need for sample cartridgeactuation.

In one embodiment, shown in FIG. 42, an imaging fiber 6070 with amicrolens 6080 (e.g., a GRIN lens) may be positioned in the opticalpathway. Light from an excitation source 3000 may be collimated with alens 3010, filtered 3020 to the appropriate wavelength, passed throughan open shutter 3030, reflected 90° by a (long pass) dichroic mirror3040, and focused onto the distal end of the fiber 6070 with a lens3010. The excitation light may travel through the fiber 6070 and excitefluorophores present in the sample. The fluorescence emission from theexcited fluorophores may be collected by the fiber's microlens 6080,transmitted through the fiber 6070, collected with a lens 3010, passedthrough a long pass dichroic mirror 3040, filtered 3050 to theappropriate wavelength(s), and imaged with a detector 3060. Thefluorescence images may then be processed and a meaningful value may bereported to an operator. This optical platform may provide more uniformillumination and an increased field of view. While the above descriptionis specific for fluorescent changes, it should be understood that thesystem may be modified to capture any kind of spectroscopic change.

In another embodiment, shown in FIG. 43, multiple optical (imaging)fibers 6070 containing microlenses 6080, may be used to imagesimultaneously multiple regions of interest in the sample, eliminatingthe need to actuate the sample cartridge 1060. At the proximal end, thefibers may be separated at fixed positions, relative to the sample. Atthe distal end, the fibers may be bundled together. In operation, thelight path is similar to previous examples, except that multiple areasof the sample are excited. The fluorescence emission from the multipleexcited sample areas is collected by the fibers 6070 and imaged 6090simultaneously with a CCD camera. The fluorescence image may beprocessed and a meaningful value may be reported to an operator. Anadvantage to using multiple optical fibers is that multiple areas can beimaged simultaneously with one image and without moving the sampleand/or cartridge. While the above description is specific forfluorescent changes, it should be understood that the system may bemodified to capture any kind of spectroscopic change.

FIG. 44A depicts a top view of a sample cartridge 1060 including aparticle-based platform 7000, a membrane platform 7010, and reagents7020 positioned on the cartridge. FIG. 44B depicts a side view of asample cartridge 1060 including a particle-based platform 7000, and amembrane platform 7010. In an embodiment, single core optical fibers6070 may be used in the optical platform to provide more uniformfluorescent signals from particle-based platforms 7000 containingfluorescent particles. At the proximal end, the fibers 6070 may beseparated at fixed positions, relative to the particle-based platform7000 (e.g., one fiber per particle, above or below). The fibers 6070 mayautomatically line up when the sample cartridge 1060 is snapped intoposition. At the distal end, the fibers 6070 may be bundled together. Inoperation, the light path may be similar to previous examples. Thefluorescence emission from the multiple excited particles may becollected by the fibers and imaged 6090 simultaneously with a CCDcamera. The fluorescence intensities may be processed and a meaningfulvalue may be reported to the operator. An advantage is that multipleparticles may be imaged simultaneously in one image, with improvedsignal uniformity and with moving the sample or cartridge. While theabove description is specific for fluorescent changes, it should beunderstood that the system may be modified to capture any kind ofspectroscopic change.

An optical platform may display images detected by a detector on acomputer. A computer coupled to the instrument may be a desktop, laptop,handheld or other computer equipped with commercial or custom software.The software may contain algorithms and/or neural networks for imageanalysis. Images may be analyzed by the computer for fluorescenceproperties, such as intensity, lifetime, spectral characteristics,polarization, absorption properties, luminescence properties, number ofparticles or some function thereof, size, shape or combination of any ofthese.

In another embodiment, an analyte detection device may include acartridge that holds a particle-based detector and/or a membrane-baseddetector. The cartridge may be a disposable cartridge and may act as thechemical and biochemical-sensing component of the analyte detectiondevice. The cartridge, which shape may be adapted to various needs, maybe composed of index-matching, molded or machined plastics, metals,glass or a combination thereof. In one embodiment, a cartridge mayinclude one or more reservoirs for holding reagents, sample, buffer,fluids for analysis of samples, and waste, that are connected via one ormore microfluidic channels and/or valves. The cartridge may include oneor more analysis and/or separation surfaces (e.g., membrane or the like)and/or sensing particles housed in a supporting array. A membranesurface may trap and/or separate particulate matter of interest (e.g.,cells, microbes, small pieces of tissue, polymer, glass or metalparticles, or conjugates thereof). The particle-based platform componentmay include sensing particles. Sensing particles may react with analytesof interest (e.g., proteins, DNA and RNA oligonucleotides, metals orother solution-phase analytes). In certain embodiments, a cartridge maybe able to detect particulate matter and/or solution-phase analytesconcurrently.

In some embodiments, particles in a sensor array may be opticallyencoded with one or more fluorophores, chromophores, etc. which may beused to identify the location of the particle in the array and/or theidentity of the analyte. Such an encoding scheme may be used in acombination membrane-particle-based cartridge and may facilitatemanufacture of the cartridge. Encoded particles may be placed in thesample for the purpose of sample or reagent identification (e.g., asample identification bar code). In one embodiment, the membrane may beused to trap the particles. Particles may identify a patient. In anembodiment, in addition to membrane-based analysis, particle-basedanalysis is performed by the instrument. Such particles may also be usedto calibrate the instrument and/or monitor flow rates.

A system for analysis of analytes is configured, in one embodiment, tosubstantially simultaneously combine the analysis of cellular andprotein material in fluids. In one embodiment, a dual function analytedetection device may include both particle- and membrane-basedplatforms, suitable for the measurement of a variety of analytessimultaneously. In one embodiment, the dual function analyte detectiondevice may be used to measure both blood proteins and count blood cells.The device may provide quick and accurate diagnosis of specificdiseases, which may save lives and lift the financial strain on both thehealthcare system and the patient.

FIG. 45 depicts one embodiment of an analyte detection device thatincludes both a particle-based detection system 7000 and amembrane-based detection system 7010. Both the particle-based detectionsystem 7000 and the membrane-based detection system 7010 are formedwithin a body 7020. Body 7020 may be formed from a thermoplasticmaterial (e.g., polydimethylsiloxane). In one embodiment, the device maybe casted in a thermoplastic material from a micromachined mold that hasbeen modified to accommodate both the particle-based and membrane-baseddetection systems. A channel 7030 may be formed between membrane-baseddetection system 7010 and the particle-based detection system 7000 andconnect the two analysis devices. A waste reservoir 7040 may also beincorporated into the body 7020 to collect liquid samples afteranalysis.

FIG. 46 depicts an exploded view of a portion of a detection systemsupport system. The device depicted in FIG. 46 may be used to support aparticle-based detection system or a membrane-based detection system.Detection system support system may be composed of multiple concentricrings. In one embodiment, the detection system support system may becomposed of four concentric rings. A structure ring 7050 may hold thewhole assembly. A compression ring 7060 may hold the membrane or aparticle based sensor array in place as it screws down into structurering 7050. The compression ring 7060 may also include a sample deliveryopening 7070 through which fluids are delivered on to the membrane orthe particle based sensor array. A reagent ring 7080 may be positionedadjacent to compression ring 7050. Reagent ring 7080 may be actuatedsuch that sample delivery may occur through reservoir holes 7090. Duringuse, reagent ring 7080 may be rotated such that reservoir holes 7090 maybe aligned with sample delivery opening 7070 of the compression ring7050. An opening (not depicted) is formed on the top surface ofcompression ring 7050 to allow fluid to flow from a reservoir hole 7090through compression ring 7060 and out of the sample delivery opening,onto the membrane or particle based sensor array, see FIGS. 45 and 46.This allows the reagents to be delivered sequentially through theoperation of an actuator system. An actuator system 8000 is depicted inFIG. 45. A window ring 8010 may hold an optical window in a place thatallows optical observation of the sample on the membrane or theparticles in the particle-based sensor array. A system of actuators maybe used to push liquid down through the system. Alternate actuatingsystems may be used that include an additional ring to push down ontoreservoirs formed in reagent ring 7080. In an embodiment, a pressure ofthe ring on the reservoir may pressurize a liquid sending the liquid tothe detection system.

In the embodiment depicted in FIG. 45, an actuator system 8000 may beused to send samples and/or regents through the membrane based detectionsystem and the particle-based detection system. During use, a sample ora reagent may be introduced into any of the reservoir holes 7090. At theappropriate times, reagent ring 7080 may be rotated to a position withone of the reservoir holes 7090 oriented over an opening in compressionring 7060. Operation of actuator 8000 may deliver fluid from reservoirhole 7090 through compression ring 7060 to the membrane-based detectionsystem 7010. After flowing through membrane-based detection system 7010,fluid that is not trapped by the membrane-based detection system mayflow into channel 7030 and to particle-based detection system 7000. Inone embodiment, a reagent ring 7080 may be customized to include variousnumbers and volumes of reagents. Reagent ring 7080 may also bepre-packaged and easily mass produced. The size and shape of reagentring 7080 may be adjusted to accommodate the different needs dictated byvarious applications.

In some embodiments, an external pumping system may be used to deliverfluids to an analyte detection device, as depicted in FIG. 47. Ananalyte detection device may include both a particle-based detectionsystem 7000 and a membrane-based detection system 7010. Both theparticle-based detection system 7000 and the membrane-based detectionsystem 7010 are formed within a body 7020. Body 7020 may be formed froma thermoplastic material (e.g., polydimethylsiloxane). In oneembodiment, the device may be casted in a thermoplastic material from amicromachined mold that has been modified to accommodate both theparticle-based and membrane-based detection systems. A first channel8020 may couple pump 8030 to particle-based detection system 7000. Asecond channel 8040 may couple pump 8030 to membrane based detectionsystem 7010. Pump 480 may allow delivery of different samples or thesame sample to each of the detection systems 8030. A third channel 8050may be formed between particle-based detection system 7000 and wastereservoir 7040. Waste reservoir 7040 may also be coupled tomembrane-based detection system 7010 to receive waste fluids.

FIG. 48 depicts an embodiment of a single-use cartridge for use in thedetection of analytes. Cartridge 1060 may be formed from a variety ofmaterials, such as polymers, glasses, or metals. In one embodiment, apolydimethylsiloxane (PDMS) casting may be used. The cartridge 1060 maybe designed to interface with a variety of peripheral fluidics systems.Alternatively, a pumpless design may be used by incorporating acustomizable number of blister packs 8060, or substantially sealedreservoirs, into the cartridge 1060. Blister packs 8060 may includedelivery fluids, reagents or other development fluids. Blister packs8060 may be coupled to a detection system 8070 through microchannels8080. Detection system 8070 may be a membrane-based detection system ora particle based detection system. Reservoir 8090 may be used to collectthe fluids from detection system 8070.

Blister packs 8060 may be used to deliver fluids to detection system8070. Various activating systems may be used to force liquid from theblister through the microchannels 8080. Applying pressure to a blisterpack may release delivery fluids, reagents, and/or other developmentfluids. Increasing pressure applied to blister pack may increase theamount of fluid delivered from the pack. In one embodiment, depicted inFIG. 49, liquid may be forced from blisters 8060 using a roller 9000.Contact of roller 9000 against blister 8060 may force liquid fromblister toward detection system 8070.

In some embodiments, a cartridge may be designed with connectors thatmay interface with standard human fluid collection devices. Theseconnectors may be designed to be compatible with a wide variety ofmicrofluidic fittings and tubings. An example of such cartridge is shownin FIG. 50. The cartridge includes two input connectors, a sampleintroduction port 9010 and a buffer port 9020. The sample introductionport 9010 may introduce samples into the cartridge. Samples introducedinto the cartridge may be conducted through channels into a mixingchamber 9030. In the mixing chamber 9030, analytes in the sample may mixwith reagents previously placed in the mixing chamber. The reagents mayinteract with the analytes in the sample to aid in visualization of theanalytes. In one embodiment, cartridge may include a microfluidic valve9040. Microfluidic valve 9040 may control a flow of the fluid throughthe cartridge. Flow of sample fluids may be directed through samplecheck window 9050 or to the membrane 9060 for detection of the analytes.Fluids passing through the membrane may be collected in waste reservoir.In an embodiment, fluids that pass through the sample check window mayalso be collected in the waste reservoir. It should be understood thatwhile the above-description refers to a membrane-based detection system,the cartridge may be readily adapted to a particle-based detectionsystem.

In one example, reagents may be stored in a lyophilized form. FIG. 51Adepicts lyophilized reagents 9070 disposed in a mixing chamber 9030.Lyophilized reagents 9070 may be mixed with the sample 9080 uponintroduction of the sample into mixing chamber 9030 of the cartridge, asdepicted in FIG. 51B. Once the chamber 9030 is filled with the sample,the mixture of the sample and reagents 9070 will flow out of the chamberto other parts of the cartridge based on the positioning of microfluidicvalves in the cartridge, as depicted in FIG. 51C.

FIGS. 52A-52C depict a series of schematic diagrams showing anembodiment of the operation of the cartridge. Valves may be actuatedelectro-mechanically and/or manually through a keypad of a readerenclosing the cartridge. Various combinations of valves and actuatorsmay be used to build various fluidics circuitries depending on thenumber and nature of the reagents needed for each application. Forexample, as depicted in FIG. 52A, the sample is introduced through thesample introduction port 9010. The microfluidic valve 9090 may be placedin an orientation that blocks flow of the sample to the detectionsystem, as depicted in FIG. 52A. Thus, as the sample exceeds thecustomizable metered volume of the mixing chamber, the sample overflowsand passes through a sample check 9050 channel and into a wastereservoir. The sample may be thus observed through an opening in thereader/cartridge assembly. After an incubation time typical of eachapplication, delivery of the sample to the membrane is actuated, afterswitching of one or more microvalves 9090, as depicted in FIG. 52B. Oncethe desired sample volume has been delivered to the flow cell, themicrovalve systems 9090 are actuated to allow passage of rinsingreagents through the membrane, as depicted in FIG. 52C.

An alternate embodiment of a cartridge is depicted in FIG. 53. Thecartridge 1060 includes a single input connector 9010 for sampleintroduction. The sample introduction port 9010 allows samples to beintroduced into the cartridge 1060. Samples introduced into thecartridge 1060 may be conducted through channels into a mixing chamber9030. In the mixing chamber, analytes in the sample may mix withreagents previously placed in the mixing chamber. The reagents mayinteract with the analytes in the sample to aid in visualization of theanalytes. In one embodiment, cartridge 1060 may include a microfluidicvalve 9090. Microfluidic valve 9090 may be used to control flow of thefluid through the cartridge 1060. Flow of the sample fluids may bedirected through sample check window 9050 or to the membrane 9060 fordetection of the analytes. Fluids passing through the membrane may becollected in waste reservoir. In one embodiment, fluids that passthrough the sample check window 9050 may also be collected in the wastereservoir. In contrast to the device depicted in FIG. 50, the cartridgemay include one or more blister packs 720. The blister packs 720 may bepressurized using either manual or automatic means to force liquid fromthe blister pack into the cartridge 1060. In an embodiment, the blisterpack 720 may include a fluid for washing the membrane-based detectionsystem (e.g., a PBS buffer solution). It should be understood that whilethe above-description refers to a membrane-based detection system, thatthe cartridge may be readily adapted to a particle-based detectionsystem.

A cartridge may include a particle-based detection system, amembrane-based detection system, or both. A cartridge may be easilycustomized to accommodate various needs. A cartridge may include acombination of valves, channels, chambers, connectors to allow easy useand access. For example, cartridges 1060 shown in FIGS. 54A and 54B maybe accommodated with an inlet, outlet, and lateral flow outlet that maybe positioned in various configurations to accommodate variousgeometries of the fluid delivery. Additionally, a cartridge may be madewith a built-in waste reservoir 10000 as depicted in FIG. 54C. The wastereservoir may be designed to handle bio-hazard materials. In anembodiment, a waste reservoir 10000 may be removable from a cartridge1060 and safely replaceable.

Multiple channels may be created in a cartridge to allow the delivery tothe detection system of a variety of reagents separately, as depicted inFIG. 55A. The reagents may be delivered to the membrane and/orparticle-based platforms 10010 of a cartridge 1060 through standard orcustomized connectors 10020. These connectors may allow delivery ofreagents to the membrane through syringes (e.g., using Luer fittings),or any standard or customized fittings to accommodate a variety of fluiddelivery devices. Reagents may be pre-packaged within the cartridge anddelivered to the detection system through capillary action or variousactuation methods. FIG. 55B depicts an embodiment of a cartridge inwhich the sample may be deposited or introduced to a chamber 10030 whereit is drawn to the membrane or particle-based platforms 10010 of acartridge 1060 through capillary action, actuation, or pump action. FIG.55C depicts an embodiment of a cartridge 1060 that may include acombination of standard or customized connectors 10020, and reagentchambers 10030 that may be actuated. This cartridge also may include a“bull's eye” window where the sample is delivered to a metered chamber.Observation of sample through the “bull's eye” indicates overfilling ofthe chamber to a waste reservoir, and readiness of the metered volume ofsample to be delivered to the membrane. FIG. 55D depicts a diagram of anembodiment of a cartridge 1060 with one or more connectors and/orchambers 10030 modified to receive a capillary collection tube 10040that includes an analyte. The capillary tube inner surface may bemodified with a blood anti-coagulant. An inner surface of the capillarytube may be coated with an antibody mixture or other chemical orbiological species used in the detection. The capillary 10040 is thenintroduced to the cartridge where the sample may be delivered to amembrane and/or a particle-based platform 10010 in the detection systemthrough capillary action, actuation, or pumps.

In some embodiments cartridges 1060 may include a trap 10050, which isused to inhibit air from flowing to the detection system, as depicted inFIG. 56A. Using a trap 10050 may release air from a sample flowing froma capillary 10040 or sample collection device to a membrane or aparticle-based platform 10010. A similar system including a built-inremovable waste reservoir 10070 is shown in FIG. 56B. The cartridgedepicted in FIG. 56B may also include a lateral flow outlet 10080 thatmay be directly coupled to the trap 10050 in order to get rid ofaccidental bubbles in the flow cell.

As is shown in FIGS. 57A-C, the cartridge flow cell may be connected toa pumping system through a variety of fluidics interfaces. The fluidicsinterface may include various types of fittings ranging from snug fittubing, snap-on, standard or customized connectors that may be madere-usable or disposable. In these examples, the fluidics interfaces maybe made complimentary to the cartridge connectors or tubes. A cartridge1060 may be coupled to a fluid bus 1090 using snug fit tubing 10090, asdepicted in FIG. 57A. A fluid bus 1090 may be coupled to a cartridge1060 such that fluid in fluid delivery lines 10100 may not leak. Duringuse, fluid may flow from fluid delivery lines 10100, through a fluid bus1090, into a membrane or a particle-based platform 10010 in a cartridge1060 and into a waste reservoir 10070. As depicted in FIG. 57B, acartridge may be coupled to fluid lines 10100 via snap-on connectors10110. A fluid bus 1090 may be sealed to a cartridge 1060 via snap-onconnectors 10110. A sample may flow through a fluid delivery line 10100into a capillary 10040 or sample reservoir and then into a bubble tap10050. Air may escape a sample via a fluid delivery line 10100 coupledto the trap 10050 and sample may flow to a membrane and/or aparticle-based platform 10010 of the cartridge 1060. Waste fluids mayflow through fluid delivery lines 10100 to a waste reservoir 10070. Acartridge may also be coupled to fluid delivery lines 10100 and/or afluid bus 1090 using customized connectors 10120, as depicted in FIG.57C. A fluid bus may be reusable. Sample may flow into a capillary 10040and into a trap 10050 and/or particle-based platform and/or membraneregions 10010 of a cartridge 1060 via fluid delivery lines 10100. Traps10050 and/or particle-based or membrane platforms 10010 may be coupledto a waste reservoir 10070, such that waste fluid from traps, membranesplatforms, and/or particle-based platforms may flow to a waste reservoirafter analysis.

Dual functional analyte detection devices (e.g., analytes detectiondevices that use both membrane-based and particle-based detectionsystems) may be used in a number of applications. In one embodiment, adual functional analyte detection device employs both particle- andmembrane-based platforms suitable for the measurement of blood proteinsand the counting of blood cells, respectively. Both platforms have beentested separately for each of their respective applications and werefound to produce excellent assay characteristics. Here, a new designmerging the two approaches/technologies is presented for the measurementof Troponin T and CRP and the counting of white blood cells from theblood of patients suffering from chest pain. On site measurement ofTroponin T (particle-based) will identify those patients that indeedsuffered a heart attack, while simultaneous measurements of CRP(particle-based) and counting of white blood cells (membrane-based) mayidentify those who have suffered a heart attack and are in need ofimmediate and aggressive therapy, such as coronary angioplasty. Thisportable Point-of-Care system may serve as the ideal instrument for thetimely diagnosis of a heart attack and provide direction for thephysician towards the appropriate treatment.

Another application for a dual functional analyte detection device isfor detecting and identifying bacteria. Typical methods of detection,used for years by microbiologists, require the growth of single bacteriainto bacterial colonies in different types of media, followed by atimely identification process involving morphological and biochemicaltests. The classification of microorganisms through conventionalmicrobiological counting and enumeration methods involves the use ofnucleic acid stains or cocktails of stains, which are capable ofdifferentiating between gram-positive and gram-negative bacteria, andbetween dead or living organisms. However, these procedures suffer frompoor specificity and are not easily adapted to online rapid analysis.This series of steps, although often providing very accurate resultsrepose on the expertise of highly trained personnel, and require lengthyand complicated analysis. Most commonly available assays for thedetection of spores or bacteria involve the use of enzyme-linkedimmunosorbent assays (ELISA), polymerase chain reaction (PCR),electrochemical transduction, optical and microarray detection,flow-through immuno-filtration, acoustic sensors, and flow cytometry.While demonstrating high specificity, reproducibility, and somecapabilities of multiplexing, these methods generally require lengthyanalysis times, and are not compatible with real-time analysis. Forexample, PCR analysis presents the most promising technological responseto an urgent need for a rapid detection method for Bacillus anthracis.However, despite the potential advantages of using PCR for thisapplication, some of the drawbacks include long analysis time, reagentcosts, and the difficulty of using PCR to detect many bacteria or sporespecies simultaneously.

In one embodiment, a dual functional analyte detection device employsboth particle- and membrane-based platforms suitable for the measurementdetection of specific bacteria. Using a dual functional analytedetection device, various types of spores and bacteria may first becaptured on the membrane for a presumptive test. This analysis willinclude gram stain, live/dead distinction, shape and size recognition,and counting. The membrane test will also be utilized in conjunctionwith an antibody stain or stain cocktail for preliminary identification.A positive signal may then trigger a series of confirmation chip-basedtests of the bacterial lysate for the detection of the protein/toxin/DNAcontent of the microbe, providing a final assessment of the nature ofthe microorganism.

Another application for a dual functional analyte detection device isfor measuring complete blood count. The complete blood count (CBC) isthe most common diagnostic test administered worldwide. It combines theanalysis of platelets, red and white blood cells, with measurements ofhemoglobin, and hematocrit. In addition to routinely providing a generalhealth evaluation, CBC is widely used as the initial screening test forthe diagnosis of a great number of diseases, as well as for monitoringdisease progression and response to treatment. A complete and moredefinitive medical diagnosis however, very often requires the additionalmeasurement of selected proteins, gases or chemicals in the bloodstream. For example, an initial visit to the doctor's office may mostlikely include a CBC, in conjunction with other tests, such as achemistry test (Ca²⁺, phosphorus, glucose), electrolytes (Na⁺, K⁺,chlorides, bicarbonate, CO₂), kidney and liver functions (blood ureanitrogen, creatinine, alanine aminotransferase, aspartateaminotransferase, bilirubin, alkaline phosphatase, gamma glutamyltranspeptidase, and lactic dehydrogenase) and others (albumin, globulin,sedimentation rate). Outside the hospital, completion of these testsvery often require multiple blood samples to be drawn and shipped foranalysis in specialized laboratories, increasing the time form whichresults will be available form hours to days. In a great number ofinstances, the output of a CBC test determines the need foradministering more specialized tests, which may require additional time,instruments and procedures. All of these delays are putting a toll onpatients, doctor's overloaded schedule, and sometimes the outcome of adisease, when these tests are barely available.

The reporting interval for an emergency CBC test can vary from minutesto hours in a hospital setting to a number of hours for routine testing,but for most patients, samples are shipped to specialized locations foranalysis, and are not available for at least a day. Hematology analyzersof FACS machines are routinely used for obtaining white blood celldifferentials. However, the chemistry panel involves a battery of testthat require various analytical tools and that are for the most partdone separately.

In one embodiment, a dual functional analyte detection device employsboth particle- and membrane-based platforms suitable for the measurementdetermination of CBC. The dual approach employs both the particle-basedplatform to measure levels of selected protein, enzymes, and chemicalsin blood and a membrane-based platform that is dedicated to the cellularanalysis of blood. The feasibility of the system with enzymes, metalcations, DNA, influenza, and hepatitis has already been shown. Analysisof blood cellular content has also been demonstrated with anti CD45stains of leukocytes in whole blood.

In some embodiments, an analyte detection device may employparticle-based analysis using membrane-based platforms to detect one ormore analytes in a fluid. This embodiment may be an alternative, or usedin combination with, an array-based platform for detecting analytes. Inan embodiment, defined populations of particles may be generated thatdetect a specific analyte. Defining populations of particles may includedefining sets of size-and/or color-coded particles according to severalmeasurable parameters.

Various types of schemes may be used to define different populations ofparticles. In an embodiment, the system may utilize, for example, purepopulations of specific sizes of particles. Particles may range fromabout 1 μm to about 100 μm, with each population of particles having aparticle size distribution within about 5 μm of the selected medianparticle size. In an embodiment, each population of size-coded particlesmay be further defined into coded subsets. Coding of particles may beaccomplished by coupling an identification molecule to the particle.Examples of identification molecules include, but are not limited tocolorimetric dyes and fluorescent dyes. Coding of particles may beaccomplished by coupling different identification molecules to differentsets of particles or by coupling varying concentrations of anidentification molecule to different sets of particles. In suchembodiments, individual populations of particles may be generated thatare well defined and are distinguishable on the basis of size,light-absorbance, intensity of light absorbance or combinations thereof.For example, in an embodiment, two populations of particles may begenerated by coupling particles of the same size to different amounts ofa red fluorescent dye. The two populations of particles may bedistinguished from each other in a mixed population of particles bycollecting digital images of the mixed population of particles andcomparing the pixel intensity of the particles in the mixed population.

In an embodiment, each defined population of particles may be chemicallysensitized to detect one analyte of interest in a mixture of analytes.This may be achieved by coupling a receptor that binds the analyte to adefined population of particles. As used herein, a receptor that iscapable of binding to the analyte may generally be referred to as a“capturing receptor.” Binding of an analyte in a fluid to a capturingreceptor may substantially remove at least a portion of the analyte fromthe fluid phase by capturing the analyte on the surface of theanalyte-sensitized particles. Examples of capturing receptors include,but are not limited to DNA, RNA, proteins, enzymes, oligopeptides,oligonucleotides, antigens, and antibodies. In some embodiments, thedefined set of particles may be dedicated to the capture and detectionof one analyte of interest. By having multiple distinct populations ofparticles, each population of particles may be configured to capture andaid in the detection of a different analyte.

In an embodiment, different populations of particles may be chemicallysensitized to detect different analytes in a mixture of analytes. Thechemically sensitive particle, in one embodiment, may be capable of bothbinding the analyte(s) of interest and creating a detectable signal. Inone embodiment, the particle creates an optical signal when bound to ananalyte of interest. In one embodiment, a detectable signal may becaused by the altering of the physical properties of an indicator ligandbound to the receptor or the polymeric resin. In one embodiment, twodifferent indicators may be attached to a receptor or the polymericresin. When an analyte is captured by the receptor, the physicaldistance between the two indicators may be altered such that a change inthe spectroscopic properties of the indicators is produced. A variety offluorescent and phosphorescent indicators may be used for this sensingscheme. This process, known as Forster energy transfer, is extremelysensitive to small changes in the distance between the indicatormolecules. In another embodiment, an indicator ligand may be preloadedonto the receptor. An analyte may then displace the indicator ligand toproduce a change in the spectroscopic properties of the particles. Inthis case, the initial background absorbance is relatively large anddecreases when the analyte is present. The indicator ligand, in oneembodiment, has a variety of spectroscopic properties that may bemeasured. These spectroscopic properties include, but are not limitedto, ultraviolet absorption, visible absorption, infrared absorption,fluorescence, and magnetic resonance. The indicator may be chosen suchthat the binding strength of the indicator to the receptor is less thanthe binding strength of the analyte to the receptor. Thus, in thepresence of an analyte, the binding of the indicator with the receptormay be disrupted, releasing the indicator from the receptor. Whenreleased, the physical properties of the indicator may be altered fromthose it exhibited when bound to the receptor. In an embodiment, theanalyte molecules in the fluid may be pretreated with an indicatorligand. Pretreatment may involve covalent attachment of an indicatorligand to the analyte molecule. After the indicator has been attached tothe analyte, the fluid may be passed over the particles. Interaction ofthe receptors on the particles with the analytes may remove the analytesfrom the solution. Since the analytes include an indicator, thespectroscopic properties of the indicator may be passed onto theparticle. By analyzing the physical properties of the sensing particlesafter passage of an analyte stream, the presence and concentration of ananalyte may be determined. As previously described, the receptor itselfmay incorporate the indicator. The binding of the analyte to thereceptor may directly lead to a modulation of the properties of theindicator. Such an approach may use a covalent attachment or strongnon-covalent binding of the indicator onto or as part of the receptor,leading to additional covalent architecture. Each and every receptor mayuse a designed signaling protocol that is unique to that receptor. In analternative embodiment, two or more indicators may be attached to theparticle. Binding between the receptor and analyte causes a change inthe communication between the indicators, again via either displacementof one or more indicators, or changes in the microenvironment around oneor more indicators. The communication between the indicators may be, butis not limited to, fluorescence resonance energy transfer, quenchingphenomenon, and/or direct binding. Further examples of methods ofproducing signals on particle that include a receptor specific for ananalyte of interest are described in U.S. Pat. No. 6,589,779 entitled“General Signaling Protocol for Chemical Receptors in ImmobilizedMatrices,” which is incorporated herein by reference.

In an embodiment, multiple analytes may be detected simultaneously usingmixed populations of analyte-specific particles, where each populationof analyte-specific particles is dedicated to the capture and detectionof one analyte of interest. In one embodiment, adding a population ofanalyte-specific particles to a fluid containing that analyte may causethe analyte to bind to the particles. Because each population ofparticles is sensitized to detect only one analyte in a fluid, thatanalyte may have limited binding to any other population of particles.

In order to detect the presence of an analyte bound to the surface of apopulation of particles, a means of visualizing surface-bound analytesis required. This may include adding a visualization agent to theanalyte-bound particles. As used herein, a “visualization agent”generally refers to an agent, such as a chemical agent, that interactswith analyte-bound particles, and allows the visualization of particlesthat have specifically bound the analyte for which they are chemicallysensitized. In an embodiment, a visualization agent may include a secondreceptor that binds to the analyte. As used herein, a second receptorthat binds to the analyte may generally be referred to as a “detectingreceptor.” Examples of detecting receptors may include, but are notlimited to DNA, RNA, proteins, enzymes, oligopeptides, oligonucleotides,antigens, and antibodies. In an embodiment, the detecting receptor maybe a polypeptide molecule that binds to the analyte. Alternatively, thedetecting receptor may include a second antibody directed against theanalyte. In one embodiment, a method of detecting multiple analytes in afluid may rely on immunological reactions that take place on the surfaceof the particles. In an embodiment, the visualization agent may beoptically distinguishable from the particles. For example, thevisualization agent may be coupled to an indicator or dye that isspectroscopically distinct from the particles. In an embodiment, thevisualization agent may be coupled to a fluorescent dye that isdistinguishable from the fluorescent or colorimetric dye that definesthe particles. In an embodiment, detecting an analyte in a fluid mayinclude detecting a first signal from the particles, and a second signalfrom the visualization agent.

In an embodiment, populations of particles with captured analytes ofinterest may be passed through a flow cell equipped with a porousmembrane, such as that which is described in detail above and depictedin FIG. 1. The analyte detection system may be configured to allow forthe delivery of a test fluid and its flow through the system, as well asthe visualization of the contents therein using an optical imagingapparatus. The use of a porous membrane may allow the particles to becaptured on the surface of the membrane while allowing the passage offluids and any compounds dissolved therein, including but not limited touncaptured analytes, unbound receptors or antibodies, test fluids,diluents, solvents, wash buffers and the like. Suitable porous membranesfor use in the embodiments presented herein would include thosemembranes with a pore size smaller than the diameter of the smallestpopulation of size-coded particles used in the assay. In an embodiment,the membrane fitted to the flow cell system may be a polycarbonate tracketched porous membrane such as, for example, a nuclepore® type membrane.

In an embodiment, detecting an analyte in a fluid may include mixing oneor more populations of analyte-specific particles with the test fluidand a detecting receptor, and passing the mixture across a porousmembrane disposed in an analyte detection device. In an embodiment, ananalyte detection device may include a flow cell system, such as thatwhich has been described in detail above. Passing theparticle-containing fluid through the membrane equipped flow cell maycause the particles to be captured on the surface of the porousmembrane. In an embodiment, the flow cell may be configured to allow forthe microscopic examination of the contents captured on the membranesurface. This may include fabricating components of the flow cell, suchas, for example, the top member 140 and bottom member 150, from amaterial that is substantially translucent to visible and/or ultravioletlight. This may facilitate the optical imaging of signals emitted fromparticles captured on the surface of the membrane using optical imagingtechniques.

In an alternate embodiment, detecting an analyte in a fluid may includepassing a test fluid through an analyte detection device equipped with aporous membrane and populations of analyte specific particles capturedthereon. In an embodiment, the analyte detection device may include aflow cell system, such as that which has been described in detail above.In this embodiment, passing the fluid through the porous membrane maycause the analyte to interact with the analyte-specific particlescaptured thereon. In an embodiment, the detecting receptor may be addedto the test fluid prior to passing the test fluid though the analytedetection system. In another embodiment, the detecting receptor may bepassed through the analyte detection system after the test fluid hasbeen passed through the system.

In an embodiment, the analyte detection system may be coupled to anoptical imaging station. The optical imaging station may include, forexample, a microscope capable of visualizing the signals emitted fromthe particles and/or capable of determining the size of the particles. Adetector may be used to capture images of the membrane-capturedparticles. A detector may include a detection device, such as a CCDdigital imaging apparatus, and analytical software that is capable ofanalyzing digital images, such as, for example, Image Pro 4.0 or thelike. Suitable optical instrumentation and imaging software platform foruse in the embodiments presented herein have been described above. Insome embodiments, the analyte detection system coupled to an opticalimaging station may provide a means for efficient capture of populationsof analyte-specific particles and the static imaging of the analytescaptured thereon.

In an embodiment, digital images of particles captured on a field of themembrane may be acquired and the signals emitting from the particles maybe analyzed. For example, in an embodiment where particle populationsare defined by red fluorescence intensity, and the detecting receptor isdefined by green fluorescence, optical imaging using a red dichroicfilter would allow the identification of the particle type and itslocation on the membrane (which may be referred to as the “particleaddress”), and optical imaging using a green dichroic filter wouldidentify particle populations that have bound to the analyte ofinterest. In an embodiment, acquired images may be processed digitally.In an embodiment, digital processing may be automated to facilitate thesimultaneous detection and analysis of multiple populations ofparticles. Conversely, in alternate embodiments, a user may define areasof the membrane to be processed further. Automated digital processing ofacquired images may allow: the rapid identification of the location ofparticles and the identification of the corresponding population towhich they belong; the identification of particle populations that arespecifically bound to an analyte; and the quantitation of the analyte inthe fluid sample. Quantitation of the analyte in the fluid sample may bedetermined by measuring the intensity of the fluorescent signal emittedfrom the detecting receptor.

FIG. 58A-B depicts populations of polystyrene particles that are definedby size and by fluorescence signal intensity. FIG. 58A shows an image ofparticles captured on a membrane according to an embodiment. In thiscase, two different populations of particles are shown. The particles inthis image are of the same size, but each population of particles iscoupled to different amounts of an internal red fluorescent dye. Thesetwo populations of particles were mixed together, captured on a membranein a flow cell and imaged optically using a red dichroic filter. FIG.58A shows a view of an embodiment where polystyrene particles of thesame size are distinguished on the basis of red fluorescence intensity.Particles of high fluorescence intensity are shown as open circles, andparticles of lower fluorescence intensity are shown as shaded circles.FIG. 58B shows a line profile analysis of the particles in the boxedarea of FIG. 58A. In this case, fluorescence intensity (measured aspixel intensity) is depicted as a function of the line profile.Confirmation that only one size of particles is present in the mixedpopulation of particles may be achieved by determining the width of eachpeak at half the maximal pixel intensity. Conversely, the presence oftwo populations of particles distinguished on the basis of fluorescencesignal intensity may be demonstrated by the presence of two peak pixelintensities.

In embodiments where both the capturing receptor and the detectingreceptor are antibodies, the method of analyte detection may be referredto as a “sandwich immunoassay.” The detecting receptor may be directedto the same epitope on the analyte as the capturing receptor.Conversely, the detecting receptor may be directed to a differentepitope on the analyte than the capturing receptor. As used herein, theterm “epitope” generally refers to a region on a molecule that isrecognized by and that binds to the antigen binding sites of anantibody. In an embodiment, the detecting receptor may be coupled to adye that distinguishes the detecting receptor from the size-and/orcolor-coded particle population. For example, in an embodiment, adetecting antibody that binds to an analyte captured by a capturingantibody on the surface of first color fluorescent particles may becoupled to a second colored fluorescent dye. In such an embodiment, apositive test for the presence of an analyte would occur when apopulation of particles appears having the first color when imagedoptically using a first color filter, and the second color when imagedusing a second color filter. Conversely, particles that have the firstcolor, but do not appear to have the second color would indicate thatthe analyte is not present in the solution. In an embodiment, theconcentration of an analyte in a solution may also be determined bymeasuring the fluorescence intensity of the second dye. In an alternateembodiment, the fluorescent dye that defines the population of particlesmay be coupled to the capturing receptor rather than being coupled tothe particles.

FIG. 59A-C schematically depicts an assay for the detection of cytokineTumor Necrosis Factor (TNF-α) in a test fluid using a particle onmembrane assay system. In FIG. 59A a sandwich-type immunocomplex betweenanalyte-sensitized particles, the analyte of interest (e.g. TNF-α), anda second analyte-specific antibody is formed. In this embodiment, apopulation of 5.6 μm polystyrene red fluorescent particles is coupled toa TNF-α-specific capturing antibody. If TNF-α is present in the testfluid, the capturing antibody coupled to the population ofTNF-α-specific particles captures it. The sandwich-type immunocomplex isformed when the detecting antibody, depicted in FIG. 59A asAlexa-488-Ab, which is also specific for TNF-α and is coupled to thegreen fluorescent dye Alexa-488, binds to the complex.

FIG. 59B depicts the process involved in performing an assay accordingto an embodiment. Initially, an immunocomplex is formed in a solutioncontaining TNF-α between the particle-coupled capturing antibody thatbinds to TNF-α, soluble TNF-α, and a detecting antibody that also bindsto TNF-α and that is coupled to alexa-488. In this embodiment, particlesbelonging to the population of particles that are sensitized to detectTNF-α are depicted as closed circles, and particles belonging topopulations of that do not detect TNF-α are depicted as open circles.This step may be referred to as the “immunoreaction” step. In anembodiment, the immunoreaction step may take place in vitro, such as ina test tube, for example. After the immunoreaction step, the fluidsample containing the immunocomplexes may be passed through amembrane-equipped flow cell and captured on the membrane equippedtherewith. In an alternate embodiment, the immunoreaction step may occuron the surface of the membrane in the flow cell. In some embodiments,wash buffers, such as for example, phosphate buffered saline, or thelike, may be passed through flow cell to remove any unbound detectionantibody, or any other soluble components that may interfere with theimaging step.

Turning now to FIG. 59C, after the TNF-α-bound particles are captured onthe membrane, the particles may be optically imaged using theappropriate combinations of dichroic filter sets. In this case, a redfluorescence signal identifies the particle population address, andgreen fluorescence signal identifies the population of particles thatare bound to TNF-α. Particles belonging to the particle population thatis defined by a different fluorescence intensity (shown as open circlesin FIG. 59B-C) are not sensitized to capture TNF-α and do not emit agreen fluorescence signal.

FIG. 60 depicts a proof of principle experiment using a particle on amembrane assay system to detect TNF-α in a fluid. In this embodiment,polystyrene particles were coated with a fluorescent red dye todesignate particle address, and were coupled to an antibody directedagainst TNF-α. The particles were then added to a fluid containing noTNF-α (top panels), or to a fluid containing 10 ng/ml TNF-α (bottompanels). A detecting antibody that is coupled to alexa-488 and is alsospecific for TNF-α was added to both immunoreactions. The fluidcontaining the particles and any analyte captured thereon was thenpassed through a membrane-equipped flow cell and captured on themembrane residing therein. Captured particles were imaged using red(panels A and C) and green (panels B and D) dichroic filters. Panel Ashows the particle address when the membrane is imaged using a reddichroic filter. However, since this sample contained no TNF-α, noimmunocomplex was formed, and hence no green signal is detected when themembrane is imaged with a green dichroic filter, as shown in panel B.The sample that contained 10 ng/ml TNF-α emits a red signal from theparticles when the membrane is imaged using a red dichroic filter asseen in panel C. In contrast to the sample that lacked TNF-α, in thiscase an immunocomplex formed between the capturing antibody, TNF-α, andthe alexa-488-coupled detecting antibody, and thus a green signal isdetected when the membrane is imaged using a green dichroic filter, asseen in panel D.

FIG. 61 depicts a dose response curve to TNF-α according to anembodiment of a particle on a membrane assay system. In this case,fluorescent red polystyrene particles sensitized to detect TNF-α wereexposed to either 0 ng/ml, 0.1 ng/ml, 1.0 ng/ml or 10 ng/ml TNF-a in atest fluid in the presence of alexa-488-coupled TNF-α-specific detectingantibody. The test fluids were then delivered to membrane-equipped flowcell, and the particles captured thereon were imaged optically by redand green fluorescence settings. In this case, the concentration ofTNF-α in the test samples was determined by measuring green pixelintensity according to an embodiment. The results obtained are plottedas average particle signal intensity as a function of TNF-αconcentration in the test fluid. Fluorescence images acquired using agreen dichroic filter are provided in the box above each date point.

Certain embodiments of the particle on membrane assay system may beparticularly suited to detecting evidence of one or more infectiousagents in fluids derived from patients or test subjects. Suitablesamples may be derived from body fluids, isolated, enriched or culturedcells, stool samples, swabs or aspirates taken from the nasopharyngeal,oral, genitourinary, or alimentary tracts, tissue homogenates, celllysates, bronchoalveolar or gastric lavage, tissue aspirates or anyother patient sample collected according to standard procedures in theart. Suitable body fluids may include, but are not limited to, wholeblood, fractionated blood, blood plasma, serum, saliva, urine, mucoussecretions, cerebrospinal fluid, lymphatic fluid, pulmonary orgastointestinal secretions or contents, semen, lacrimal secretions orcombinations thereof. Non-limiting examples of infectious agents thatmay be detected according to some embodiments may include, viruses,bacteria, parasites, fungi, yeasts, prions, or combinations thereof.

In an embodiment, the particle on membrane assay system may be used todetect and diagnose viral infections and diseases caused by viruses.Examples of viral infections and diseases caused by viruses that may bediagnosed according to some embodiments may include, but are not limitedto, retroviruses, human immunodeficiency virus (HIV), AcquiredImmunodeficiency Syndrome (AIDS), hepatitis viruses, adenovirus,poliovirus, Epstein-Barr virus, mononucleosis, cytomegalovirus,influenza, viral encephalitis, viral meningitis, varicella-zoster virus,herpes simplex viruses, chickenpox, smallpox, Coxsackie virus,enteroviruses, Dengue fever, coronavirus, Severe Acute RespiratorySyndrome (SARS), Ebola, viral hemorrhagic fevers, measles, flaviviruses,yellow fever, paramyxoviruses, West-Nile virus, rabies, or any othervirus or viral disease for which natural, synthetic or recombinantpolypeptide or nucleic acid capturing and detecting receptors may beavailable.

In an embodiment, viral particles may be detected in a test fluid bycoupling virus-specific receptors or antibodies to particles. Suitablereceptors or antibodies may include, but are not limited to, receptorsor antibodies that recognize and bind to viral coat proteins andglycoproteins, capsid proteins, structural proteins, nucleoplasmicproteins, viral enzymes such as, for example, viral polymerases, viralintegrases, or the like. Detecting receptors may includeindicator-coupled receptors or antibodies.

In an alternate embodiment, viruses may be detected in a test fluid bycoupling nucleic acids, such as DNA or RNA, whose nucleic acid sequencesare complementary to and hybridize with at least a portion of the viralgenome. In these embodiments, detecting receptors may include enzyme,chromophore or fluorophore-coupled nucleic acids whose nucleic acidsequences are homologous to and hybridize with the same or withdifferent portions of the viral genome as the capturing receptor orproteins that bind to sequences within the viral genome. Embodiments inwhich nucleic acids are employed as capturing receptors may be usedeither alone or in combination with other nucleic acid hybridization oramplification techniques commonly used in the art, such as, for example,PCR.

In some cases, infectious agents, such as viruses, may be present atlevels too low to be detected directly. In such cases, it may bepreferable to detect antibodies that are specific for an infectiousagent, and that may be present in test fluids derived from patients ortest subjects. In such embodiments, a positive test for an infectiousagent would include a positive test for the presence of antibodiesspecific for the infectious agent. In an embodiment, a purified orrecombinant polypeptide molecule, or a synthetic oligopeptide, orderivatives or combinations thereof, whose polypeptide sequencesubstantially corresponds to at least a portion of the polypeptidesequence of a protein that is expressed by an infectious agent, may becoupled to a population of particles and function as capturing receptor.The particles may then be mixed with a test fluid derived from a patientor test subject. If the patient or test subject has been exposed to theinfectious agent, or is infected with the infectious agent, and hasmounted at least an humoral immune response against the infectiousagent, then antibodies present in the test fluid would bind to theirrespective epitopes on the capturing receptor. The particles may then bepassed though an analyte detection device and captured on a porousmembrane, according to an embodiment. In an embodiment, anindicator-coupled detecting receptor that recognizes and binds toantibodies may be used to detect antibodies that are bound to particles.Suitable detecting receptors that bind specifically to antibodies arewell known in the art and may include, but are not limited to,antibodies whose epitopes are the heavy or light chains of antibodies(e.g. anti-IgG, anti-IgE, anti-IgA, anti-IgD or anti-IgM antibodies),Staphylococcus protein A, Streptococcus protein G, chimeric protein AG,complement proteins, recombinant or purified FcR immunoglobulinreceptors, or the like.

In an embodiment, the particle on membrane assay system may be used todetect and diagnose HIV infection. Populations of particles may becoupled to HIV proteins and used to detect antibodies specific to HIVthat may be present in a body fluid derived from a patient suspected ofbeing seropositive. Suitable HIV proteins that may be used include, butare not limited to, HIV coat proteins and glycoproteins, capsidproteins, structural proteins, nucleoplasmic proteins, viral enzymes, orthe like. Non-limiting examples of HIV proteins that may be suitable foruse in the embodiments presented herein include the HIV gag proteinsp53, p24, p17, p7, p6, p2 or p1, the HIV env glycoproteins gp120, gp41or gpl 60, HIV enzymes including integrase (p31), reverse transcriptase(p51 or p66), RNase H (p 15), protease (p10), the HIV nef proteins(p25/p27), the HIV vif protein p23, HIV rev protein p19, HIV vpr protein(p12/p10), HIV vpu protein (p16) or HIV tat proteins (p16/p14). Theseembodiments may include coupling the fill-length protein or derivatives,portions or combinations thereof to particles. Antibodies to multipleHIV proteins may be detected simultaneously in a patient sampleaccording to an embodiment. By testing for antibodies to multiple HIVproteins present in a single sample, the likelihood of a false positiveresult may be reduced.

In an embodiment, performing an HIV test on a test fluid by detectingHIV specific antibodies may include mixing one or more populations ofparticles coupled to HIV proteins with the test fluid. Suitable testfluids may include fluids containing blood or serum, saliva, urine orany other fluid or body fluid described previously. In an embodiment,the mixture may be passed across a porous membrane disposed in ananalyte detection device, and the particles in the mixture capturedthereon. In an embodiment, excess or residual test fluid may beevacuated from the flow cell device by flushing the chamber with anappropriate volume of wash buffer. In an embodiment, anindicator-coupled detecting antibody such as, for example, anAlexa-488-coupled anti-human IgG antibody may be provided to thechamber. Optical imaging and analysis of the membrane-captured particlesmay then proceed according to embodiments described above.

In a further embodiment, HIV virions or proteins may be detected influid samples, tissue homogenates or cell lysates. In an embodiment,antibodies that recognize HIV proteins may be used as capturingantibodies to perform a sandwich immunoassay as described in detailabove. Detecting antibodies may be specific for the same or differentHIV proteins as the capturing antibodies. For example, HIV virions maybe detected in a fluid by coupling a capturing antibody whose epitope isone or more regions of the HIV env protein gp120. In this embodiment, asuitable detecting antibody may include the same antibody as thecapturing antibody that is coupled to an indicator rather than toparticles. Alternatively, the detecting antibody may include anindicator-coupled antibody whose epitope is a different region of gp120.In yet further embodiments, a capturing antibody may include anindicator-coupled antibody that binds to an epitope on a differentprotein such as, for example, p24 or gp41. In yet another embodiment,capturing and detecting receptors that may be used to detect HIV virionsmay include those cellular receptors that bind to HIV proteins.Non-limiting examples of cellular receptors that bind to HIV proteinsmay include, for example, CD4, chemokine receptors CCR5 or CXCR4, orcombinations thereof.

In some embodiments, an instrument may include one or more disposablecartridges. Such an instrument may portable. In some embodiments, acartridge may be designed such that the cartridge is removablypositionable in an instrument. A cartridge may include one or moredetection systems. Light from an optical platform of the instrument maypass onto a detection region and a detector in the optical platform mayacquire images (e.g., visual or fluorescent) of the sample, and/or ofsample-modulated particles.

FIG. 62 depicts an embodiment of a cartridge. FIG. 63 depicts anembodiment of a portion of the cartridge of FIG. 63. A cartridge 10130may include a sample collection device 10140, as depicted in FIGS. 62and 63. A sample may be delivered to the sample collection device 10140.In an embodiment, a sample collection device may include a samplepick-up pad. A sample may be introduced into the sample collectiondevice. In one embodiment, a sample may be introduced into a samplecollection device using a syringe or a pipette. Alternately, a samplemay be introduced from a person directly to the sample collectiondevice. For example, human blood may be introduced by forming a smallincision in portion of a human body. The portion of the human body maybe brought close to the sample pick-up pad such that blood flows fromthe incision in the human body to the sample pick-up pad.

Sample from the sample collection device 10140 may flow into one or moremicrofluidic channels 10150 coupled to the sample collection device.Capillary action may allow a sample to flow into a channel. A valve10190 may restrict flow of sample from the sample collection device10140. A valve 10190 proximate a sample collection device 10140 and avalve 10200 proximate an overflow reservoir 10210 in channel 10150 maybe opened such that a predetermined amount of sample may be measured.During use the sample flows into channel 10150 until it fills thechannel. The channel may hold a predetermined amount of fluid. An amountof sample greater than the predetermined amount may flow through valve10200 into an overflow reservoir 10210. After a predetermined amount ofsample is measured in channel 10150, valve 10190 and valve 10200 may beclosed. Closing a valve 10190 proximate a sample reservoir may inhibitsample greater than a predetermined amount from flowing towards adetection region 10180. Closing a valve 10200 proximate an overflowreservoir 10210 may inhibit the predetermined amount of sample fromflowing towards the overflow reservoir.

In some embodiments, a reservoir 10160 containing buffer and/or reagentsmay be coupled to a channel 10150. Fluid from the reservoir 10160 maypush the predetermined sample towards a detection region. A buffer maybe released from a buffer reservoir 10160 coupled by a channel to thechannel containing the sample. In one embodiment, a buffer may bereleased from a reservoir 10160 by an actuator. Fluid from a reservoirmay push the sample towards a mixing region or a detection region. Asample may mix and/or react with the fluid in a mixing region prior toflowing to a detection region. In certain embodiments, a reagent pick uppad 10170 may be positioned on a cartridge 10130 such that fluid from areservoir 10160 may be able to flow over the reagent pick-up pad towardsthe detection region 10180. As depicted in FIG. 64, fluid from areservoir 10160 may transfer reagents on a reagent pick-up pad 10170into channel 10150. In some embodiments, reagents may be in a dehydratedor lyophilized state. Fluid from the reservoir may reconstitute andtransfer the reagents as the fluid passes over the regent pick up pad10170. Fluid from the reservoir 10160 containing reagents may be coupledto a detection region 10180 through a channel 10150. Detection regionmay include a particle based sensor array or a membrane-based system.Fluids in the cartridge 10130 may be collected in a waste reservoir10190 after flowing past a detection region 10180, as depicted in FIG.62. By containing all fluids within the cartridge, a user's exposure toreagents and sample may be substantially minimized.

In some embodiments, one or more reagents may be contained in areservoir positioned on a cartridge. A reagent reservoir may include ablister pack, as depicted in FIG. 65A. FIG. 65B depicts across-sectional view of an embodiment of a blister pack. A blister packmay include one or more reagents in a sealed reservoir. A sealedreservoir may substantially contain reagents in the reservoir untilneeded. Pressure applied to a blister pack may break one or moresurfaces of the blister pack such that reagent is released from theblister pack. In an embodiment, a blister of a blister pack may beformed of a first material 10220 and a second materials 10230, where asecond material is configured to rupture or break prior to the firstmaterial when pressure is applied to the blister. In an embodiment, ablister may include a first material configured not to break whenpressure is applied to a blister and a second material configured tobreak when pressure is applied to a blister. A blister may be made ofpolyvinyl chloride (PVC); polyvinylidene chloride (PVDC); polyethylene(PE); polypropylene (PP); polyacrylonitrile (PAN); cyclic olefincopolymer (COC); fluoropolymer films; foil such as aluminum foil orplastic foil; and/or combinations thereof. A wall of a blister may beformed of layers of polypropylene, cyclic olefin copolymer. For example,a blister wall may be formed from a layer of cyclic olefin copolymer inbetween two layers of polypropylene. A wall of a blister may be formedof layers of polypropylene, cyclic olefin copolymer, andpolyacrylonitrile. In an embodiment, a wall of a blister may be formedof layers of polyvinyl chloride, cyclic olefin copolymer, andpolyvinylidene chloride.

In some embodiments, one or more valves may be coupled to channels inthe cartridge. FIG. 66 depicts an embodiment of valve placement inchannels on a cartridge. Valves may direct flow of a fluid through achannel. One or more valves coupled to microfluidic channels 10150 mayallow a predetermined amount of sample from a sample reservoir 10140 tobe analyzed. In one embodiment, a cartridge 10130 may include a firstvalve 10152 which may allow control of the introduction of sample into aportion of channel 10150. A first valve 10152 may be closed duringsample collection to inhibit sample from flowing towards the detectionregion. A first valve 10152 may be opened to allow a predeterminedamount of sample to flow into a microfluidic channel 10150 coupled tothe detection region 10180. One or more other valves in the cartridgemay be closed to direct a flow of sample in the cartridge.

In certain embodiments, a predetermined amount of sample may be measuredinto channel 10150. In one embodiment, sample is introduced into channel10150 by opening of valve 10152. Sample is block from detection region10180 by closing of valve 10156. As sample fills channel 10150, apredetermined amount of sample may be collected by allowing sampleexceeding the predetermined amount to enter an overflow reservoir orregion. A second valve 10154 proximate an overflow region may be openedas sample enters channel 10150 to allow sample exceeding thepredetermined amount to flow into an overflow region and/or wastereservoir 10190. After a predetermined amount of sample is measured in achannel 10150, first valve 10152 and second valve 10154 are closed toprevent sample from the sample collection region and the overflow regionfrom flowing to a detection region 10180. A third valve 10156 may beopened to allow a sample to flow towards a detection region 10180. Afourth valve 10158 may be opened to allow buffer from a buffer reservoir10160 to push the measured sample towards the detection region 10180.One or more valves in a fifth set of valves 10159 may be opened to allowone or more reagents to flow towards a mixing chamber and/or detectionregion 10180. One or more reagent reservoirs 10160 may be actuated suchthat reagent may flow to the detection region. Reagents may mix with asample in a mixing chamber and/or mixing region. Reagents from a reagentreservoir 10160 may flow over one or more reagent pick-up pads 10170 andreconstitute one or more reagents on the reagent pick-up pad. In oneembodiment, a buffer solution may be passed over a reagent pick-up padand flow towards a mixing region and/or detection region 10180. A samplemay be analyzed in a detection region, such as a particle-based or amembrane-based detection region and/or platform. A cartridge may beflushed during or after analysis by buffer from one or more reservoirscontained in the cartridge. Fluids may flow from a detection region to awaste reservoir.

Valves may include valves configured for microfluidic channels, such asgate valves, check valves, passive microvalves, and/or pinch valves. Inone embodiment, pinch valves may be used in a cartridge to control flowin microfluidic channels. Fluids such as a sample, reagents, and/orbuffers may flow through channels in a cartridge and valves may controlthe direction of the flow. A pinch valve may include an opening 10240 ina cartridge, as depicted in FIG. 67A. A channel 10150 may be accessedthrough the opening 10240. The opening may have a concave lower surface10250. When a cartridge is loaded in an instrument, openings 10240 inthe cartridge may be aligned with actuators 10260 coupled to theinstrument.

In some embodiments, an actuator 10260 may be positioned in an opening10240 of a cartridge above a channel 10150 after a cartridge ispositioned in an instrument, as depicted in FIG. 67B. A lower surface10250 of the opening 10240 may have a shape such that a bottom surfaceof an actuator 10260 fits in the lower surface of the opening. Asdepicted in FIG. 67C, an actuator 10260 may apply pressure on thechannel 10150 such that fluid is inhibited from flowing through thechannel. When pressure is applied to the channel 10150 to restrict flowthrough the channel, the valve is closed. In an embodiment, a lowersurface 10250 of the opening may have a depth substantially equal to thediameter of the channel exposed in the opening. FIG. 68 depicts across-sectional view of an embodiment of a pinch valve in a cartridge. Apinch valve may include an opening 10240 in a cartridge 10130 thatallows access to a channel 10150. A channel 10150 may be positionedabove a lower surface 10250 of the opening 10240.

FIG. 69A depicts an exploded view of an embodiment of a cartridge. Acartridge may include a top seal layer 10270, a top microchannel layer10280, a center layer 10290, a bottom microchannel layer 10300, and/or abottom seal layer 10310. Layers of a cartridge may be coupled together.Layers of a cartridge may be sealed together. Creating a cartridge fromseveral layers may facilitate fabrication. A top seal layer 10270 mayinclude access 10320 to a sample collection device 10140 or samplecollection pick-up pad. Top 10280 and/or bottom 10300 microchannellayers may create a system of microchannels through the cartridge. Acenter layer 10290 may include reservoirs 10160 containing buffer and/orreagents, a portion of a sample collection device 10140, and/or a wastereservoir 10190. FIG. 69B depicts a top view of an embodiment of acartridge. FIG. 69C depicts a perspective view of an embodiment of acartridge. FIG. 69D depicts a bottom view of an embodiment of acartridge.

FIG. 70 depicts an exploded side view of an embodiment of a cartridge.Top 10380 and bottom 10450 seal layers may substantially contain fluidin the top 10390 and bottom 10430 microchannel layers. In an embodimenta fluid may flow from a top microchannel layer 10390 through a detectionregion 10410 in the center layer 10400 to a bottom microchannel layer10430. Fluid may flow through the bottom microchannel layer 10430 to awaste reservoir.

FIG. 71 depicts a side view of an embodiment of a cartridge 10130. Insome embodiments, fluid may flow from a top microchannel layer 10390through a detection region 10410. Fluid may pass from the detectionregion 10410 through the bottom microchannel layer 10430 to a wastereservoir. Top 10380 and bottom 10450 seal layers may substantiallyretain fluid in microchannel layers.

FIG. 72A depicts an exploded view of another embodiment of a cartridge.An opening 10320 in the top seal layer 10270 may allow sample to bedeposited in a sample collection device 10140 on the cartridge. When asample is deposited in the cartridge one or more valves in a channel10150 may inhibit a sample from flowing towards a detection region10180. FIG. 72B depicts an embodiment of an arrangement of valves priorto and during deposition of a sample on the cartridge. During depositionof a sample, first 10330, second 10340, third 10350, and fourth 10360valves may be closed to inhibit flow of sample through the cartridge.

In some embodiments, after a sample is deposited on the cartridge, anamount of sample may flow from the sample collection device 10140through a channel 10150 via capillary action, as depicted in FIG. 73A.FIG. 73B depicts an arrangement of valves that allows sample to flowinto a channel. A first valve 10330 may be open to allow a sample toflow into a microchannel. Second 10340 and third 10350 valves may beclosed to control a flow of the sample. Closing a second valve 10340 mayinhibit sample from flowing towards a buffer reservoir. Closing a thirdvalve 10350 may allow a predetermined amount of sample to be measured. Afourth valve may be opened to allow sample in the channel to flow intoan overflow reservoir.

FIG. 74A depicts an embodiment of sample flow in a cartridge. In someembodiments, it may be desirable to allow a portion of sample to flowover a detection region 10180. A predetermined amount of sample 10145may be measured and allowed to flow towards the detection region 10180.A predetermined amount of sample may be measured by allowing sample inexcess of a predetermined amount to flow into an overflow region 10210.An overflow region 10210 may be coupled to a waste reservoir 10190.Valves in the cartridge may inhibit sample in a main channel fromflowing into channels coupled to reservoirs 10160. After a predeterminedamount of sample is measured, valves may be closed to inhibit additionalsample from flowing into the channel containing the predetermined amountof sample. For example, as depicted in FIG. 74B, a first valve 10330 maybe closed to inhibit additional sample from a sample collection device10140 from entering a channel. Second 10340 and third 10350 valves mayremain closed. A fourth valve 10360 may be closed to prevent sample fromthe overflow region 10210 from flowing into the channel.

After a predetermined amount of sample is measured, a reservoir 10160may be actuated, as depicted in FIG. 75A. A reservoir may contain bufferand/or reagents. An actuator may release buffer from a reservoir. Abuffer reservoir may be similar to a blister pack. As depicted in FIG.75B, a third valve 10350 may be opened to allow fluid to flow towards adetection region. Actuation a buffer reservoir 10160 may cause buffer tobe released from a reservoir into a microchannel. A reservoir 10160 maybe coupled to the cartridge so that fluid from the reservoir may flowfrom the reservoir towards the detection region 10180. A reservoir 10160may be positioned in the cartridge so that buffer from a reservoir maypush a predetermined amount of sample 10145 towards a detection region10180. In an embodiment, a buffer may flow from a reservoir 10160 over amembrane in a detection region 10180 to wash the membrane after thesample flows over the membrane. The buffer may then pass over themembrane and into the waste reservoir 10190.

FIG. 75B depicts an arrangement of valves in an embodiment of acartridge that may allow a buffer to push a sample through amicrochannel and towards a detection region. A first valve 10330 may beclosed so that a sample may be inhibited from reentering a samplecollection device 10140 or sample pick-up pad. A second valve 10340 maybe opened to allow fluid from a buffer reservoir to flow towards adetection region. A fourth valve 10360 may be closed such that fluid maybe inhibited from flowing into the overflow reservoir 10210. A thirdvalve 10350 may be open such that fluid may flow towards a detectionregion.

As the reservoir 10160 is actuated, buffer is released into a channel10150 that couples the reservoir to a main channel containing themeasured sample 10145. A main channel may couple a sample collectiondevice 10140 to a detection region 10180 and/or waste reservoir 10190.The released buffer may push the predetermined amount or measured amountof sample 10145 towards a detection region 10180, as depicted in FIG.76. Sample may pass over a detection region 10180, such as a membrane,and into a waste reservoir 10190. As depicted in FIG. 77, a bufferreservoir 10160 may be activated and buffer may be released such thatthe substantially all of the measured amount of sample and/or bufferflows over the detection region 10180. Fluid (e.g., sample and/orbuffer) that passes through the detection region 10180 may flow into awaste reservoir 10190.

A reservoir 10370 containing reagents and/or buffer may be actuated torelease reagents and/or buffer into channels in the cartridge, asdepicted in FIG. 78A. FIG. 78B depicts an embodiment of valves in acartridge. A first valve 10330 may be closed to prevent fluids fromentering a sample collection device 10140. A second valve 10340 may beclosed after buffer is released from a reservoir to push sample towardsa detection region. Third 10350 and fourth 10360 valves may be closed tosubstantially inhibit fluid from flowing into an overflow region 10210and/or away from a detection region. A fifth valve 10360 proximate areservoir 10160 containing buffer and/or reagents may be opened to allowbuffer and/or reagents to flow over a detection region.

Actuating a reservoir 10370 may push fluids from a reservoir over areagent pad towards a detection region 10180 and/or waste reservoir10190. A reservoir 10370 may include buffer and/or reagents. Reagents ona reagent pack may be reconstituted as the fluid from the reservoir10370 passes over the reagent pack. A reservoir 10370 may be coupled toa detection region 10180 and/or a waste reservoir 10190 via one or morechannels. One or more reagents may react with the sample in thedetection region. In some embodiments, reagents from one or more reagentreservoirs and/or reagent packs may mix with a sample in a mixingchamber. After a fluid containing reagents from a reagent pad and/or areservoir 10370 pass over a detection region 10180. Reagents may reactwith a portion of the sample in the detection region 10180. Unreactedreagents, excess reagents, and/or buffer may flow from the detectionregion and into a waste reservoir 10190. A reservoir 10370 may beactuated until a predetermined amount of reagents and/or buffer passover the detection region 10180 and into a waste reservoir 10190. Insome embodiments, a reservoir may be actuated to push buffer from thereservoir over the detection region. In certain embodiments, afteranalysis of the detection region, a reservoir may be actuated to releasebuffer and wash the detection region. Analysis of the sample may berepeated after analysis of the detection region.

In this patent, certain U.S. patents and U.S. patent applications havebeen incorporated by reference. The text of such U.S. patents and U.S.patent applications is, however, only incorporated by reference to theextent that no conflict exists between such text and the otherstatements and drawings set forth herein. In the event of such conflict,then any such conflicting text in such incorporated by reference U.S.patents and U.S. patent applications is specifically not incorporated byreference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention may be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description to theinvention. Changes can be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims. In addition, it is to be understood that featuresdescribed herein independently may, in certain embodiments, be combined.

1. A method of detecting an analyte in a fluid comprising: forming a mixture of one or more particles with the fluid, wherein each particle comprises a receptor configured to interact with the analyte, and wherein particles that interact with the analyte undergo a detectable spectroscopic change; passing the mixture of particles and fluid across a porous membrane disposed in an analyte detection device, wherein the analyte detection device is configured to capture the particles on the porous membrane, wherein the analyte detection device comprises: a porous membrane; a top member positioned at a spaced distance above the porous membrane such that a first cavity is formed between the top member and the porous membrane, wherein the top member covers at least a portion of the porous membrane, and wherein the top member is substantially transparent to light, and a bottom member positioned below the porous membrane, wherein the bottom member is configured to receive fluid flowing through the porous membrane during use; detecting spectroscopic signals produced by particles captured on the porous membrane; and determining if the analyte is present based on the detected spectroscopic signals.
 2. The method of claim 1, wherein each of the particles comprises an identification molecule, wherein the identification molecule coupled to each particle is specific for the analyte that interacts with the particle, the method further comprising determining if the analyte is present by determining the identity of the identification molecule on particles in which a spectroscopic signal has been detected.
 3. The method of claim 1, wherein each of the particles comprises a predetermined size, wherein the size of each particle is specific for the analyte that interacts with the particle, the method further comprising determining if the analyte is present by determining the size of the particles in which a spectroscopic signal has been detected.
 4. (canceled)
 5. The method of claim 1, further comprising detecting additional analytes, wherein each analyte is detected by one or more particles that are configured to produce a spectroscopic signal when one or more particles interact with the additional analytes.
 6. The method of claim 1, wherein forming a mixture of one or more particles with the fluid further comprises adding a detecting receptor to the fluid, wherein the particles are configured to produce a first signal, and wherein the detecting receptor is configured to produce a second signal, and wherein determining if the analyte is present comprises detecting the first and second signals. 7-17. (canceled)
 18. The method of claim 16, wherein the analyte detection device further comprises a membrane support in contact with the membrane, and wherein the membrane support comprises pores that allow fluid to flow through the membrane support at a speed that is equal to or greater than the speed that fluid passes through the porous membrane.
 19. The method of claim 16, wherein the analyte detection device further comprises a membrane support in contact with the membrane, and wherein the membrane support provides sufficient support of the membrane to inhibit sagging of the porous membrane.
 20. (canceled)
 21. The method of claim 1, wherein the top member comprises a fluid inlet configured to allow fluid to be introduced to the membrane through the top member.
 22. The method of claim 1, wherein the bottom member comprises a fluid outlet configured to allow fluid to pass from the membrane out of the analyte detection device. 23-27. (canceled)
 28. The method of claim 1, wherein the receptor comprises an antibody that binds to at least a portion of a virus.
 29. (canceled)
 30. The method of claim 1, wherein the receptor comprises an antibody that binds to at least a portion of the human immunodeficiency virus. 31-35. (canceled)
 36. The method of claim 1, wherein the receptor comprises an antibody to the analyte.
 37. The method of claim 1, further comprising adding a visualization agent to the particles captured on the porous membrane.
 38. (canceled)
 39. The method of claim 1, further comprising passing a background fluid through the analyte detection device and detecting an image captured on the porous membrane prior to passing the fluid containing the analyte across the porous membrane.
 40. The method of claim 1, further comprising washing the surface of the porous membrane. 41-46. (canceled) 